BIOLOGICAL OVERVIEW The neurogenetic role of lethal of scute resembles that of the other three proneural gene members in the achaete-scute complex (achaete, scute and asense). This overview will examine instead the role of lethal of scute in the specification of muscle progenitors.

Muscle development takes place in two phases. First, the pattern of muscle development is laid down by allocation of founder cells, specific cells in the mesoderm, each one serving as a founder for a unique muscle. Second, founder cells recruit neighboring myoblasts to form the syncylial precursors of mature muscle by fusion. Initially l'sc expression is widespread in cell clusters, but becomes allocated to muscle founder cells through the action of the Notch pathway.

The expression of lethal of scute in mesoderm is transient, occuring in twist expressing cells. From late stage 9 until stage 12, there are at least 19 clusters of l'sc expressing cells in each hemisegment. In each cluster, one cell accumulates higher levels of l'sc than the other cells in the cluster. It is this single cell, allocated from a cluster of cells, that moves to a position close to the ectoderm and eventually becomes a muscle founder cell.

Genes coding for three transcription factors, (nautilus, S59 and msh1) are each expressed in small groups of cells destined to differentiate into muscle cells. Another transcription factor, MEF2, is required for myosin expression and the fusion of myoblasts. None of these are selector genes initiating muscle fate. Genes with a decisive role in myogenesis, similar to members of the MYO-D family in vertebrates, have not been found in Drosophila.

In neurogenic mutants, the domains of mesodermal S59 expression are expanded. This suggests the Notch pathway is involved in restricting the expression of l'sc and S59 to single cells, the same way it functions in neuroblast differentiation. l'sc is not the only factor involved in founder cell specification. In some instances no l'sc is found in the founder cell, and l'sc mutation does not completely upset muscle specification.

Founder cell specification is easily comparable to neuroblast specification. The role of l'sc in muscle specification is analagous to the role of achaete-scute genes in neural growth. Specification of muscle founder cells is one of the many different processes involving the Notch pathway, a significant proportion of which involve proneural genes (Carmena, 1994).

Determination of cell fate along the anteroposterior axis of the Drosophila ventral midline

The Drosophila ventral midline has proven to be a useful model for understanding the function of central organizers during neurogenesis. The midline is similar to the vertebrate floor plate, in that it plays an essential role in cell fate determination in the lateral CNS and also, later, in axon pathfinding. Despite the importance of the midline, the specification of midline cell fates is still not well understood. This study shows that most midline cells are determined not at the precursor cell stage, but as daughter cells. After the precursors divide, a combination of repression by Wingless and activation by Hedgehog induces expression of the proneural gene lethal of scute in the most anterior midline daughter cells of the neighbouring posterior segment. Hedgehog and Lethal of scute activate Engrailed in these anterior cells. Engrailed-positive midline cells develop into ventral unpaired median (VUM) neurons and the median neuroblast (MNB). Engrailed-negative midline cells develop into unpaired median interneurons (UMI), MP1 interneurons and midline glia (Bossing, 2006).

The determination of midline cells appears to take place during germband elongation, since by germband retraction most midline subsets can be identified by the expression of unique molecules. The anteroposterior position of midline siblings was determined during germband elongation. Midline precursors were labelled with the lipophilic dye DiD or DiI in embryos expressing GFP in the Engrailed domain (en-GAL4/UAS-tauGFP). After division of the precursors, the daughter cells were followed throughout development, recording their segmental position at stage 10 and stage 11. MP1 interneurons, UMI and MNB neurons each arise from one precursor, and their daughter cells occupy fixed anteroposterior positions during germband elongation. The four daughter cells of the two glial precursors can be located either in the middle of the segment or just anterior to the Engrailed domain. VUM neurons arise from three midline precursors, and the six daughter cells of these precursors are located inside the Engrailed domain and immediately posterior to the domain, in the anterior of the next segment (Bossing, 2006).

In summary, the midline glia and MP1 interneurons are the most anterior midline subsets, followed by a second pair of midline glia and a pair of UMIs, and, finally, the VUM and MNB neurons. DiI labelling cannot resolve whether the MP1 interneurons or the midline glia are the most anterior cells. Since determination of the MP1 interneurons depends on Notch/Delta signalling, it is possible that the anteroposterior position of the most anterior midline cells, the midline glia and MP1 interneurons, is random. Interestingly, four VUM neurons and the MNB neurons seem to arise from the anterior compartment of the next posterior segment. These cells initiate Engrailed expression half-way through germband elongation, and, during germband retraction, they join the adjacent anterior segment to become the most posterior midline subsets (Bossing, 2006).

The separation of midline cells into two compartments is an early and crucial step in midline cell determination. During germband elongation, a second phase of Engrailed expression is initiated at the midline in the anterior cells of the next posterior segment. During germband retraction, these cells join the anterior segment where they develop into posterior midline cells. Expression of late Engrailed depends on Hedgehog signalling and the proneural gene lethal of scute. Lethal of scute precedes Engrailed expression and is also activated by Hedgehog. Hedgehog and Wingless signalling counteract each other to define the position of the Lethal of scute cluster, and to divide the 16 midline daughter cells into eight non-Engrailed- and eight Engrailed-expressing cells (Bossing, 2006).

It has generally been believed that the determination of the different subsets of midline cells occurs before the precursors undergo their simultaneous division at stage 8. This view is challenged by the observation that expression of the proneural gene lethal of scute, and the subsequent expression of Engrailed, is initiated in midline daughter cells at stage 10, about one hour after the precursors divide. In the neuroectoderm, proneural genes confer neural competence to a cluster of ectodermal cells. Lateral inhibition by Notch/Delta signalling then limits the expression of proneural genes to a single cell, which delaminates from the ectoderm and becomes a neural precursor (neuroblast). Because the only neuroblast at the ventral midline (median neuroblast, MNB) originates from the proneural Lethal of scute cluster, it seems likely that the MNB is selected by lateral inhibition from a cluster of midline daughter cells. However, the process of lateral inhibition in the midline differs from that in the adjacent neuroectoderm. In the neuroectoderm, a single cell delaminates and the remaining cells of the cluster cease proneural expression and give rise to the epidermis. The proneural cluster in the midline consists of three pairs of siblings generated by the division of three separate precursors. Labelling of single precursors shows that, during the selection of the MNB, only one of the two labelled siblings enlarges, but both delaminate from the embryo. In contrast to the neuroectoderm, the remaining cells of the midline cluster continue to express Lethal of scute after delamination of the MNB. This extended proneural expression might be necessary to maintain neural competence in the non-delaminating cells that develop into VUM neurons (Bossing, 2006).

The results cannot exclude the possibility that some of the midline subsets are determined as precursors, but at least two of the five midline subsets, the VUM neurons and the MNB, are determined after precursor cell division. There are striking similarities between the development of the ventral midline of Drosophila and grasshopper embryos. In grasshopper, Engrailed expression can be detected in the MNB, its progeny and the midline precursors MP4 to MP6, which each give rise to two neurons with projection patterns comparable to the Drosophila VUM neurons. Hence, the same types of midline cells express Engrailed in grasshopper and Drosophila, but in grasshopper Engrailed expression is initiated in all midline precursors prior to division (Bossing, 2006).

In the ectoderm from stage 10 onwards, Wingless, Engrailed and Hedgehog maintain the expression of one another by a feedback loop: Wingless maintains Engrailed expression, Engrailed is needed for the expression of Hedgehog and Hedgehog maintains Wingless expression. In the developing CNS, Wingless and Hedgehog expression seem to be independent of each other. At the ventral midline there are two separate stages of Engrailed expression: the early phase is maintained by Wingless; the late phase does not require Wingless and is instead activated at stage 10 by Hedgehog signalling and Lethal of scute. In the ectoderm, Wingless and Hedgehog act in concert to maintain Engrailed expression, but at the midline Wingless and Hedgehog act in opposition: Wingless represses and Hedgehog activates Lethal of scute expression (Bossing, 2006).

Wingless may repress Lethal of scute expression indirectly, via its maintenance of early Engrailed. As in the ectoderm, midline Engrailed represses expression of the Hedgehog receptor Patched and the Hedgehog signal transducer Cubitus interruptus. It is possible that early Engrailed-expressing midline cells are not able to receive the Hedgehog signal. However, ectopic expression of Hedgehog is able to induce Lethal of scute in all midline cells, suggesting that Wingless may repress Lethal of scute by a yet unknown mechanism. Recently it has been reported that a vertebrate wingless orthologue, Wnt2b, can maintain the naïve state of retinal progenitors by attenuating the expression of proneural and neurogenic genes (Bossing, 2006).

The differentiation of midline cells was studyed in wingless and hedgehog mutants. Consistent with earlier reports, many midline cells become apoptotic in both mutants. The surviving midline cells are not integrated into the CNS and show no morphological differentiation. The reduction in the number of Engrailed-positive midline cells in hedgehog mutant embryos may be mainly due to the loss of midline cell identity. In hedgehog mutants, midline cells lose the expression of Sim, the master regulator of midline development. As described for sim mutants, the loss of midline identity results in increased cell death and misspecification of the surviving midline cells as ectoderm (Bossing, 2006).

Ectopic expression of Hedgehog in the neuroectoderm and the developing CNS induces the expression of Lethal of scute and, approximately 40 minutes later, the expression of late Engrailed in all midline cells. It seems likely that Lethal of scute is an early target of Hedgehog signalling, and its activation may only require release from repression by the short form of Cubitus interruptus. By contrast, the delay in induction of late Engrailed in the same midline cells indicates that Engrailed activation may not only require release from repression, but also activation by the long form of Cubitus interruptus (Bossing, 2006).

Uniformly high levels of ectopic Hedgehog prevent the differentiation of most midline subsets and cause increased cell death. A single source of ectopic Hedgehog, achieved by cell transplantation, does not result in midline cell death, but reveals that the differentiation of the MP1 interneurons is more sensitive to Hedgehog levels than is the differentiation of midline glia. No other midline subsets are affected. It seems likely that Hedgehog not only activates Lethal of scute and late Engrailed, but also acts as a morphogen to control the differentiation of the MP1 neurons and midline glia (Bossing, 2006).

The phenotypes caused by ectopic Hedgehog are due to the induction of Engrailed in all midline cells. Expression of ectopic Hedgehog and ectopic Engrailed blocks the differentiation of midline glia and MP1 interneurons, and also prevents the formation of the anterior commissure. Labelling single midline precursors enabled examination of cell fates in embryos expressing ectopic Engrailed in the midline. The frequency of clones obtained indicates that ectopic Engrailed expression does not transform non-Engrailed-expressing midline subsets (MP1 interneurons, midline glia and UMI) into Engrailed-expressing subsets (VUM and MNB). Instead, embryos expressing midline Engrailed show increased cell death. In particular, the MP1 interneurons seem to be affected and were never obtained during this analysis. The low frequency of midline glia also points to apoptosis caused by expression of Engrailed. Surviving midline glia are not able to differentiate properly and cannot enwrap the remaining, posterior, commissure. All other midline subsets, including the UMIs, are able to differentiate, but they show a variety of axonal pathfinding defects that may result from the loss of anterior midline subsets and the absence of the anterior commissure (Bossing, 2006).

It is likely that genes other than hedgehog and wingless are crucial for midline cell determination. In these experiments, non-Engrailed-expressing midline subsets are never transformed into Engrailed-expressing subsets, or vice versa. gooseberry-distal may be one of these genes. From the blastoderm stage, Gooseberry-distal is expressed by two midline precursors and their four daughter cells. During early embryogenesis Gooseberry-distal expression at the midline does not depend on Wingless and Hedgehog. The anterior Gooseberry-distal cells also express Wingless and most likely give rise to the UMIs. The posterior Gooseberry-distal pair also express early Engrailed and Hedgehog, and develop into the most anterior VUM neurons. At stage 10, Hedgehog activates the expression of Lethal of scute and Engrailed in midline cells posterior to the Gooseberry-distal domain. Lateral inhibition by Notch/Delta signalling selects one cell from the Lethal of scute cluster to become the MNB. The remaining cells become VUM neurons. At stage 10, the absence of Engrailed in the six midline cells anterior to the Gooseberry-distal domain defines a cell cluster that will give rise to midline glia and MP1 interneurons. Based on the expression of Odd, Delta mutants have an increased number of MP1 interneurons, up to six per segment. In Notch mutants, midline glial-specific markers are absent and the number of cells expressing a neuronal marker increases. Therefore, Notch/Delta signalling appears to determine midline glial versus MP1 interneuron cell fates in the anterior cluster. In the current model, midline cell determination takes place mainly after the division of the precursors. Although the initial determination of midline cells appears to be directed by a small number of genes, a far larger number is needed to control the differentiation of the various midline subsets. This work, and the recent identification of more than 200 genes expressed in midline cells, is the beginning of a comprehensive understanding of the differentiation of the ventral midline (Bossing, 2006).

Dual role for Drosophila lethal of scute in CNS midline precursor formation and dopaminergic neuron and motoneuron cell fate

Dopaminergic neurons play important behavioral roles in locomotion, reward and aggression. The Drosophila H-cell is a dopaminergic neuron that resides at the midline of the ventral nerve cord. Both the H-cell and the glutamatergic H-cell sib are the asymmetric progeny of the MP3 midline precursor cell. H-cell sib cell fate is dependent on Notch signaling, whereas H-cell fate is Notch independent. Genetic analysis of genes that could potentially regulate H-cell fate revealed that the lethal of scute [l(1)sc], tailup and SoxNeuro transcription factor genes act together to control H-cell gene expression. The l(1)sc bHLH gene is required for all H-cell-specific gene transcription, whereas tailup acts in parallel to l(1)sc and controls genes involved in dopamine metabolism. SoxNeuro functions downstream of l(1)sc and controls expression of a peptide neurotransmitter receptor gene. The role of l(1)sc may be more widespread, as a l(1)sc mutant shows reductions in gene expression in non-midline dopaminergic neurons. In addition, l(1)sc mutant embryos possess defects in the formation of MP4-6 midline precursor and the median neuroblast stem cell, revealing a proneural role for l(1)sc in midline cells. The Notch-dependent progeny of MP4-6 are the mVUM motoneurons, and these cells also require l(1)sc for mVUM-specific gene expression. Thus, l(1)sc plays an important regulatory role in both neurogenesis and specifying dopaminergic neuron and motoneuron identities (Stagg, 2011).

In insects, dopaminergic neurons are found in both the nerve cord and brain. One of the best-characterized insect dopaminergic neurons is the H-cell (named for its 'H'-like axonal projections), which is present in the CNS midline cells of the nerve cord. The H-cell was first described in grasshopper as one of the two progeny of the Midline Precursor 3 (MP3) cell, and shown in the moth Manduca sexta to be dopaminergic. The H-cell midline interneuron is also present in Drosophila, and similar to other dopaminergic neurons expresses a set of genes encoding dopamine biosynthetic enzymes, including pale (ple; which encodes tyrosine hydroxylase) and dopa decarboxylase (Ddc). The H-cell also expresses a vesicular monoamine transporter (Vmat), dopamine membrane transporter (DAT) and neurotransmitter receptors that receive input for serotonin (5-HT1A), glutamate (Glu-RI) and neuropeptide F (NPFR1). This characteristic pattern of gene expression and its 'H' axonal projection, to a large degree, constitute the unique character of the H-cell (Stagg, 2011).

Recent work has provided insight into the origins of midline neurons and glia (see Formation of midline precursors (MPs) and MP neurons in Drosophila). Around the time of gastrulation, the single-minded midline master regulatory gene activates the midline developmental program, and soon after 3 MP equivalence groups (MP1, MP3, MP4) of five or six cells/each form. Notch signaling selects one cell from the MP1 group to become an MP1 and the others become midline glia (MG). The same occurs for the MP3 group, with one cell becoming an MP3 and the others MG. Development of the MP4 group is more complex, with sequential Notch-dependent formation of MP4 followed by MP5, MP6 and the median neuroblast (MNB). Each MP undergoes a single division that leads to two neurons. For MP3-6, this involves binary cell fate decisions: MP3 gives rise to the dopaminergic H-cell and glutamatergic H-cell sib interneurons, and MP4-6 each gives rise to a GABAergic iVUM interneuron and glutamatergic/octopaminergic mVUM motoneuron pair. The differences in MP3-6 neuron cell fate are due to the asymmetric localization of the Numb protein, which is high in H-cell and mVUMs, but low in H-cell sib and iVUMs, and differential Sanpodo localization. Although Notch signaling directs H-cell sib and iVUMs to their fates, it is blocked in H-cell and mVUMs due to the presence of Numb. Thus, H-cell sib and iVUM cell fate and gene expression are dependent on Notch signaling, and a different regulatory program governs H-cell and mVUM fates (Stagg, 2011).

This paper asks the question: what regulatory proteins govern Notch-independent H-cell and mVUM fate and gene expression? Also addressed is how the two types of midline precursors, MPs and MNB, form. Proneural genes of the bHLH transcription family have been implicated in controlling neural precursor formation and neuron-specific transcription in both vertebrates and invertebrates. The Drosophila bHLH proneural genes, achaete (ac), scute (sc), lethal of scute [l(1)sc] and atonal have been implicated in the formation of either sensory cell or CNS neuroblast precursors. Proneural bHLH genes can also direct the formation of specific neuronal cell types, as exemplified by studies in the vertebrate spinal cord. Neuronal cell type specification is commonly due to the combinatorial action of proneural and homeodomain-containing proteins. This study demonstrates that three transcription factors: the L(1)sc bHLH protein, Tailup (Tup; Islet) Lim-homeodomain protein and the Sox family protein SoxNeuro (SoxN), work together to control overlapping aspects of H-cell gene expression. In addition, l(1)sc regulates mVUM motoneuron gene expression. All three proneural members of the Drosophila achaete-scute complex (AS-C) [ac, l(1)sc and sc] are expressed in MPs in distinct patterns, and l(1)sc is required for the formation of MP4-6 and the MNB. Thus, l(1)sc controls both midline precursor formation and, in combination with SoxN and tup, controls H-cell-specific gene expression and cell fate. Both the l(1)sc and tup genes may also function together more broadly and control non-midline dopaminergic neuron gene expression (Stagg, 2011).

The formation of midline neural precursors (five MPs and the MNB) is a dynamic, yet stereotyped process. The MPs undergo cellular changes in which their nuclei delaminate from an apical position within the ectoderm and move to the basal (internal) surface. There they divide after orienting their spindles. The precursors arise in a distinct order: MP4/MP3>MP5>MP1>MP6>MNB (Wheeler, 2008). The l(1)sc gene is required for the formation of the MP4-6 and MNB precursors and their neuronal progeny. Since MP4 could not be definitively distinguished from MP5 in Df(1)sc-B57, there is some uncertainty whether both cell types are regulated by l(1)sc. However, as most segments only possess two VUMs, and those are VUM6s in over 60% of segments, it is likely that both MP4 and MP5 are commonly affected in Df(1)sc-B57, in addition to MP6. The ac and sc genes are both expressed in MPs and MNB, yet do not appear to play a significant role in MP and MNB formation. Although l(1)sc is the major proneural gene that controls formation of embryonic neuroblasts, relatively little is known about how it functions and the identity of relevant target genes. In one study, it was shown that morphological changes that accompany neuroblast formation were dependent on l(1)sc function. This is likely to be the case for l(1)sc and MP4-6 and MNB development, as MP4-6 and MNB delamination or division was commonly absent in Df(1)sc-B57. One key question is what activates or maintains l(1)sc expression in MP3-6 and MNB? Signaling by hedgehog (hh) is likely to be important, as no midline l(1)sc expression is present in hh mutant embryos (Stagg, 2011).

Although all MPs and MNB express l(1)sc, only MP4-6 and MNB were affected in mutants - formation of MP1 and MP3 were unaffected. These differences are unlikely to be solely due to different levels of L(1)sc protein or to a combination of Ac, L(1)sc and Sc. L(1)sc protein levels are relatively constant among all five MPs and MNB, both L(1)sc and Sc are present in all MPs and MNB, and Ac, L(1)sc and Sc are present in MP1 as well as MP5,6 and MNB, yet no defects in MP1 and MP3 delamination or cell division were observed. Instead, the ability of l(1)sc to direct development of some MPs and not others may reflect the different cell states (and distinct co-factors) of the precursor populations from which each MP arises. Similarly, l(1)sc controls expression of different genes in the H-cell compared with mVUMs, probably based on their different origins (MP3 versus MP4-6). Variability in the genetic control of midline MP formation extends to the non-midline MP2 cells. The MP2s require both ac and sc for MP formation and differentiation, whereas l(1)sc does not play a role. Thus, MP2 and midline MPs (MP4-6) each require AS-C gene activity for proneural and differentiation functions, but use different AS-C family members (Stagg, 2011).

At least two distinct genetic programs control H-cell gene expression: (1) H-cell-specific gene expression is controlled by l(sc), tup and SoxN, and (2) unknown factors control gene expression that is present in both the H-cell and H-cell sib. All H-cell-specific gene expression requires l(1)sc function. tup acts in parallel to control important aspects of H-cell gene expression, including the DAT, ddc and ple genes. SoxN acts downstream of l(1)sc to control NPFR1 expression. H-cell neural function gene expression begins at stage 13, well after l(1)sc expression is absent, indicating that l(1)sc is unlikely to directly regulate these genes. However, Tup is present after stage 13 and could directly regulate DAT, ddc and ple; SoxN is also present and could directly regulate NPFR1. The l(1)sc gene regulates mVUM gene expression in a manner similar to its control of H-cell expression, but does so independently of tup, which is not expressed in mVUMs. It is noted that L(1)sc protein is present at higher levels in H-cell than mVUMs, although the significance of this is unclear. Expression of genes common to both H-cell and H-cell sib cells, including 5-HT1A, Glu-RI and tup, were not affected in l(1)sc or Notch pathway mutants, indicating a second distinct regulatory pathway. This was also observed for genes expressed in common between mVUMs and iVUMs (Stagg, 2011).

The relationship between l(1)sc and tup in controlling H-cell-specific gene expression is complex. Both genes are initially expressed in the H-cell and H-cell sib after MP3 division, but expression of both is soon restricted to the H-cell. Misexpression of l(1)sc resulted in the ectopic expression of tup in the H-cell sib, similar to other H-cell-specific genes. However, in l(1)sc mutants, tup expression was not absent in the H-cell, but instead tup expression remained present in the H-cell and sometimes in two cells: one was the H-cell and the other was (probably) the H-cell sib. In addition, l(1)sc expression was not affected in tup mutants. These results indicated that: (1) l(1)sc and tup act in parallel in the H-cell to regulate dopaminergic pathway gene transcription; and (2) l(1)sc downregulates tup in the H-cell sib, indicating a role for l(1)sc in H-cell sib development. The best marker for the H-cell sib is CG13565, although it is expressed in wild type in only 54% of segments. In Df(1)sc-B57 mutant embryos, CG13565 was expressed in 46% of segments, similar to wild type. However, given its variability of gene expression in Df(1)sc-B57 mutants and the normal variability of CG13565 expression, it remains possible that l(1)sc (and tup) may play roles in H-cell sib development. Additional experiments are necessary to determine how l(1)sc and tup function together to control H-cell-specific gene expression (Stagg, 2011).

Within midline cells, l(1)sc plays important roles in controlling H-cell and mVUM gene expression, while playing relatively insignificant roles in MP1, H-cell sib and iVUM neuronal gene expression. Whether non-midline neuronal gene regulation is regulated by l(1)sc is currently being addressed. Significantly, Df(1)sc-B57 mutant embryos show a strong reduction in DAT and ple expression in the non-midline dorsal lateral dopaminergic neurons. It has been shown that ple expression in these cells is also reduced in tup mutant embryos. Although more detailed cellular and genetic studies are required to bolster these observations, these data raise the possibility that both l(1)sc and tup may regulate gene expression in both midline and non-midline dopaminergic neurons. More generally, l(1)sc control of neuron-specific gene expression is likely to be uncommon. This is based on the observation that in the developing CNS, there is little L(1)sc protein colocalizing with newly divided Elav⁺ neurons or GMCs (Stagg, 2011).

Because of the key neurobiological and medical importance of dopaminergic neurons, there has been intensive analysis of the regulatory factors that control their development in vertebrates and C. elegans. Are the regulatory programs involved in dopaminergic neuron differentiation conserved between insects, worms, and mammals? The two key regulatory proteins that control Drosophila H-cell dopamine differentiation are l(1)sc and tup. In vertebrates the bHLH genes mouse achaete-scute homolog [Mash1; homolog of l(1)sc] and neurogenin 2 (Ngn2) play roles in midbrain dopaminergic neuron development, although the role of Mash1 is secondary to Ngn2, which has a key function in dopaminergic differentiation. However, Mash1 (as well as Ngn2) can initiate neurogenic programs of other neuronal cell types. This was emphatically demonstrated in recent work in which forced expression of Mash1 and two other transcription factor genes converted murine fibroblast cells to neurons. The mammalian orthologs of Drosophila tup, Isl1 and Isl2, play important roles in motoneuron differentiation, but have not been reported to influence dopaminergic neuron development and gene expression. Recently, C. elegans and vertebrate ETS family transcription factor genes were shown to directly regulate dopamine pathway gene expression. It will be important to identify the transcription factors in Drosophila that directly regulate dopaminergic neural function genes and connect them to the regulatory genes identified in this paper (Stagg, 2011).

cDNA clone length - 1067

Bases in 5' UTR - 46

Exons - one

Bases in 3' UTR - 268

PROTEIN STRUCTURE

Amino Acids - 257

Structural Domains

Lethal of scute has a basic helix-loop-helix domain, and an C-terminal acidic domain (Cabrera, 1988 and Martin-Bermudo, 1993). A PEST domain is located just C-terminal of the bHLH domain. Deletion of the N terminal amino acids up to the basic domain, or deletion of the C terminal domain including the PEST domain, still leaves a functional protein (Hinz, 1994).

Evolutionary Homologies

The lin-32 gene of C. elegans codes for an Achaete-Scute homolog, sufficient for specification of neuroblast fate (Zhao, 1995). Chicken Achaete-Scute homolog (CASH-1) is one element in a multiple parallel pathway involving notochord or floor plate-derived signals for the specification and development of chick sympathetic neurons (Groves, 1995). A Xenopus Achaete-Scute homolog, XASH-3, when dimerized with the promiscuous binding partner XE12, specifically activates the expression of neural genes in naive ectoderm (Ferreiro, 1994). Xenopus Achaete-Scute homologs XASH-1a and XASH-1b appear in defined regions of the developing central nervous system. The pattern of expression of the Xenopus genes is modified by the cyclops mutant (Allende, 1994).

The study of achaete-scute (ac/sc) genes is a paradigm to understand the evolution and development of the arthropod nervous system. The ac/sc genes have been identified in the coleopteran insect species Tribolium castaneum. Two Tribolium ac/sc genes have been identified -- 1) a proneural achaete-scute homolog (Tc-ASH) and 2) asense (Tc-ase), a neural precursor gene that reside in a gene complex. These genes reside 55 kb apart from each other and thus define the Tribolium ac/sc complex. Focusing on the embryonic central nervous system it is found that Tc ASH is expressed in all neural precursors and the proneural clusters from which they segregate. Through RNAi and misexpression studies it has been shown that Tc-ASH is necessary for neural precursor formation in Tribolium and sufficient for neural precursor formation in Drosophila. Comparison of the function of the Drosophila and Tribolium proneural ac/sc genes suggests that in the Drosophila lineage these genes have maintained their ancestral function in neural precursor formation and have acquired a new role in the fate specification of individual neural precursors. These studies, however, do not support a role for Tc-ASH in specifying the individual fate of neural precursors, suggesting that the ability of ac and sc to separately regulate this process may represent a recent evolutionary specialization within the Diptera. Furthermore, it is found that Tc-ase is expressed in all neural precursors, suggesting an important and conserved role for asense genes in insect nervous system development. This analysis of the Tribolium ac/sc genes indicates significant plasticity in gene number, expression and function, and implicates these modifications in the evolution of arthropod neural development (Wheeler, 2003).

The work presented in this paper together with studies on ac/sc gene function in Drosophila provide strong evidence that serial duplications of proneural ac/sc genes in the dipteran lineage led to the diversification of proneural ac/sc gene function in Drosophila. In Drosophila, ac and sc carry out functions distinct from l'sc in specifying the individual fate of the MP2 precursor. Tc-ASH can function in Drosophila as a proneural gene but like Drosophila l'sc fails to specify efficiently the MP2 fate in the CNS. Together these results suggest the ability of ac and sc to specify MP2 fate in Drosophila arose after the divergence of Drosophila and Tribolium. These data provide an example whereby a subset of duplicated genes has evolved a new genetic function while the entire set of duplicate genes has retained the ancestral function (Wheeler, 2003).

In addition to functional changes, the generation of multiple proneural ac/sc genes in the insects was paralleled by modifications to the expression profiles of these genes. In Anopheles (a basal dipteran), and Tribolium a single proneural ac/sc gene is expressed in all CNS proneural clusters. In more derived Diptera the presence of multiple ac/sc genes allows for more complex proneural ac/sc gene expression patterns. For example, Ceratitis contains two proneural ac/sc genes, l'sc and sc; l'sc is expressed in all CNS proneural clusters while sc is expressed in a subset of these clusters. In Drosophila, ac and sc are expressed in the identical pattern of proneural clusters and their expression is largely complementary to that of l'sc. The sum of proneural ac/sc expression in each species then marks all CNS proneural clusters despite differences in the expression pattern of individual proneural ac/sc genes. Thus, in Drosophila, the complete expression pattern of proneural ac/sc genes is divided between the largely complementary expression profiles of ac and sc relative to l'sc. The division of labor between proneural ac/sc genes in Drosophila has resulted in mutually exclusive expression patterns for ac and sc relative to l'sc in proneural clusters like MP2. This spatial separation of proneural gene expression probably facilitated the potential for ac and sc to acquire developmental functions distinct from l'sc (Wheeler, 2003).

Together this work and that of others on arthropod ac/sc genes highlights the utility of studying ac/sc genes in elucidating the genetic basis of the development and evolution of arthropod nervous system pattern. These studies illustrate the dynamic nature of ac/sc gene number, expression and function over a relatively short evolutionary time. Based on this, future work on ac/sc genes in additional arthropod species should continue to provide insight into the molecular basis of the evolution of arthropod nervous system development (Wheeler, 2003).

REGULATION

Promoter Structure

Thirty nucleotides upstream from the start of transcription is an unconventional TATA box (TATTTAAA). Seven CANNTG motifs (E-boxes), putitive binding sites for bHLH transcription factors, are located in this upstream region 200 to 1000 bp above the start site. These local upstream elements could function in l'sc autoregulation or activation by Achaete or Scute (Martin-Bermudo, 1993).

Transcriptional Regulation

Snail represses l'sc transcription in the presumptive embryonic mesoderm (Kosman, 1991). Elements regulating l'sc are scattered throughout 75 kb between achaete and asense. These elements activate l'sc in specific proneural clusters and as a consequence, also in their corresponding neuroblasts (Martin-Bermudo, 1993).

Short gastrulation prevents Decapentaplegic from suppressing neurogenesis laterally in the blastoderm embryo. It is possible to exacerbate defects in sog mutants by increasing the level of DPP. The earliest neuroectodermal marker affected in sog mutants with a double dose of dpp is rhomboid, which is normally expressed in lateral stripes 8-10 cells wide in wild-type embryos but rapidly narrows to stripes 4-6 cells across in sog mutants with elevated DPP. Similarly l'sc expression is reduced in sog mutants with elevated DPP. A striking feature of the affects of DPP on neural suppression and dorsalization is that neuronal suppression is induced by a lower threshold of DPP activity than is dorsalization. Much less DPP is required to suppress expression of neuroectodermal genes than is required to activate dorsal markers. For example, brief submaximal heat induction of heat shock dpp in a wild type sog background leads to nearly maximal suppression of lethal of scute, scratch and snail expression during germ band extension, but there is no detectable ectopic expression of zerknüllt in the neuroectoderm (Biehs, 1996).

The segmented portion of the Drosophila embryonic central nervous system develops from a bilaterally symmetrical, segmentally reiterated array of 30 unique neural stem cells, called neuroblasts. The first 15 neuroblasts form about 30-60 minutes after gastrulation in two sequential waves of neuroblast segregation and are arranged in three dorsoventral columns and four anteroposterior rows per hemisegment. Each neuroblast acquires a unique identity, based on gene expression and the unique and nearly invariant cell lineage that this expression produces. Little is known as to the control of neuroblast identity along the DV axis. The Drosophila Egfr receptor (Egfr) has been shown to promote the formation, patterning and individual fate specification of early forming neuroblasts along the DV axis. Molecular markers identify particular neuroectodermal domains, composed of neuroblast clusters or single neuroblasts, and show that in Egfr mutant embryos (1) intermediate column neuroblasts do not form; (2) medial column neuroblasts often acquire identities inappropriate for their position, while (3) lateral neuroblasts develop normally. Active Egfr signaling occurs in the regions from which the medial and intermediate neuroblasts will later delaminate. The concomitant loss of rhomboid and vein yields CNS phenotypes indistinguishable from Egfr mutant embryos, even though loss of either gene alone yields minor CNS phenotypes. These results demonstrate that Egfr plays a critical role during neuroblast formation, patterning and specification along the DV axis within the developing Drosophila embryonic CNS (Skeath, 1998).

In a screen to identify mutations that disrupt embryonic CNS development, one P element mutation, l(2)03033, was identified that causes a loss of essentially all Eve-positive RP2/RP2 sib neurons. This P element maps to cytological position 57F1-2 in the right arm of the second chromosome and is known to be inserted within the Egfr locus. To verify that lesions in Egfr result in a nearly complete loss of RP2 motoneurons, three additional Egfr mutants were obtained, including the Egfr null allele, flb 1K35. Essentially all Eve-positive RP2 motoneurons are absent from embryos homozygous mutant for each Egfr allele (Skeath, 1998).

The first phase of CNS development, as gastrulation commences, involves the activation of the Ac-S proneural genes in a precise pattern of proneural clusters. To investigate whether Egfr regulates As-C expression in the neuroectoderm, the expression patterns of the achaete (ac) and lethal of scute (l’sc) genes were followed in Egfr mutant embryos. Loss of Egfr causes specific defects to the DV registration of ac and l’sc gene expression in the neuroectoderm; however, no defects to the AP registration for either ac or l’sc gene expression were found. In wild-type embryos during stages 8/9, ac is expressed in the medial and lateral, but not intermediate, clusters of rows 3 and 7; l’sc is expressed in the medial and lateral, but not intermediate, clusters of row 7 and in the medial, intermediate and lateral clusters of rows 1 and 5. A single neuroblast subsequently forms from each proneural cluster. In Egfr mutant embryos, ac gene expression expands into the intermediate column in rows 3 and 7 and l’sc expression expands into the intermediate column in row 7; l’sc is expressed normally in rows 1 and 5. The lateral limits of ac and l’sc gene expression in the neuroectoderm are unaltered in Egfr mutant embryos. The changes to the DV registration of ac and l’sc gene expression in Egfr mutant embryos suggest that neuroectodermal cells in the intermediate column change their fate. Both ac and l’sc are normally expressed in the medial and lateral columns in the affected rows, thus the phenotype is consistent with intermediate cells acquiring either a lateral or a medial fate. msh-1, which is expressed exclusively in the lateral column, expands into the intermediate column in Egfr mutant embryos. In this context, it appears that ac and l’sc expression expand from the lateral column into the intermediate column in the absence of Egfr (Skeath, 1998).

The maternal Dorsal nuclear gradient initiates the differentiation of the mesoderm, neurogenic ectoderm and dorsal ectoderm in the precellular Drosophila embryo. Each tissue is subsequently subdivided into multiple cell types during gastrulation. This study investigates the formation of the mesectoderm within the ventral-most region of the neurogenic ectoderm. Previous studies suggest that the Dorsal gradient works in concert with Notch signaling to specify the mesectoderm through the activation of the regulatory gene sim within single lines of cells that straddle the presumptive mesoderm. This model was confirmed by misexpressing a constitutively activated form of the Notch receptor, Notch^IC, in transgenic embryos using the eve stripe2 enhancer. The Notch^IC stripe induces ectopic expression of sim in the neurogenic ectoderm where there are low levels of the Dorsal gradient. sim is not activated in the ventral mesoderm, due to inhibition by the localized zinc-finger Snail repressor, which is selectively expressed in the ventral mesoderm. Additional studies suggest that the Snail repressor can also stimulate Notch signaling. A stripe2-snail transgene appears to induce Notch signaling in 'naïve' embryos that contain low uniform levels of Dorsal. It is suggested that these dual activities of Snail -- repression of Notch target genes and stimulation of Notch signaling -- help define precise lines of sim expression within the neurogenic ectoderm. It is proposed that Snail functions as a gradient repressor to restrict Notch signaling. In precellular embryos, the initial snail expression pattern is broad and extends into the future mesectoderm. During cellularization, the pattern is refined and snail expression is lost in the mesectoderm and restricted to the mesoderm. The early, broad snail pattern might create a broad domain of potential Notch signaling by repressing components of the Notch pathway, such as Delta and lethal of scute. After cellularization, Notch signaling is blocked in the presumptive mesoderm by sustained, high levels of the Snail repressor. However, Notch can be activated in the mesectoderm because of the loss of Notch inhibitors repressed by transient expression of the Snail repressor. According to this model, the dynamic snail expression pattern determines both the timing and limits of Notch signaling (Cowden, 2002).

Drosophila optic lobe neuroblasts triggered by a wave of proneural gene expression that is negatively regulated by JAK/STAT

Neural stem cells called neuroblasts (NBs) generate a variety of neuronal and glial cells in the central nervous system of the Drosophila embryo. These NBs, few in number, are selected from a field of neuroepithelial (NE) cells. In the optic lobe of the third instar larva, all NE cells of the outer optic anlage (OOA) develop into either NBs that generate the medulla neurons or lamina neuron precursors of the adult visual system. The number of lamina and medulla neurons must be precisely regulated because photoreceptor neurons project their axons directly to corresponding lamina or medulla neurons. This study shows that expression of the proneural protein Lethal of scute [L(1)sc] signals the transition of NE cells to NBs in the OOA. L(1)sc expression is transient, progressing in a synchronized and ordered 'proneural wave' that sweeps toward more lateral NEs. l(1)sc expression is sufficient to induce NBs and is necessary for timely onset of NB differentiation. Thus, proneural wave precedes and induces transition of NE cells to NBs. Unpaired (Upd), the ligand for the JAK/STAT signaling pathway, is expressed in the most lateral NE cells. JAK/STAT signaling negatively regulates proneural wave progression and controls the number of NBs in the optic lobe. These findings suggest that NBs might be balanced with the number of lamina neurons by JAK/STAT regulation of proneural wave progression, thereby providing the developmental basis for the formation of a precise topographic map in the visual center (Yasugi, 2008).

Coordinated sequential action of EGFR and Notch signaling pathways regulates proneural wave progression in the Drosophila optic lobe.

During neurogenesis in the medulla of the Drosophila optic lobe, neuroepithelial cells are programmed to differentiate into neuroblasts at the medial edge of the developing optic lobe. The wave of differentiation progresses synchronously in a row of cells from medial to the lateral regions of the optic lobe, sweeping across the entire neuroepithelial sheet; it is preceded by the transient expression of the proneural gene lethal of scute [l(1)sc] and is thus called the proneural wave. This study found that the epidermal growth factor receptor (EGFR) signaling pathway promotes proneural wave progression. EGFR signaling is activated in neuroepithelial cells and induces l(1)sc expression. EGFR activation is regulated by transient expression of Rhomboid (Rho), which is required for the maturation of the EGF ligand Spitz. Rho expression is also regulated by the EGFR signal. The transient and spatially restricted expression of Rho generates sequential activation of EGFR signaling and assures the directional progression of the differentiation wave. This study also provides new insights into the role of Notch signaling. Expression of the Notch ligand Delta is induced by EGFR, and Notch signaling prolongs the proneural state. Notch signaling activity is downregulated by its own feedback mechanism that permits cells at proneural states to subsequently develop into neuroblasts. Thus, coordinated sequential action of the EGFR and Notch signaling pathways causes the proneural wave to progress and induce neuroblast formation in a precisely ordered manner (Yasugi, 2010).

Loss of EGFR function in progenitor cells caused failure of L(1)sc expression and differentiation into neuroblasts (see A model of progression of the proneural wave). In addition, elevated EGFR signaling resulted in faster proneural wave progression and induced earlier neuroblast differentiation. The activation of the EGFR signal is regulated by a transient expression of Rho, which cleaves membrane-associated Spi to generate secreted active Spi. This study also demonstrated that Rho expression itself depends on EGFR function, and thus the sequential induction of the EGFR signal progresses the proneural wave. Clones of cells mutant for pnt were not recovered unless Minute was employed, suggesting that the EGFR pathway is required for the proliferation of neuroepithelial cells. However, the progression of the proneural wave is not regulated by the proliferation rate per se (Yasugi, 2010).

The function of the Notch signaling pathway in neurogenesis is known as the lateral inhibition. A revision of this notion has recently been proposed for mouse neurogenesis, in which levels of the Notch signal oscillate in neural progenitor cells during early stages of embryogenesis, and thus no cell maintains a constant level of the signal. The oscillation depends mainly on a short lifetime and negative-feedback regulation of the Notch effecter protein Hes1, a homolog of Drosophila E(spl). This prevents precocious neuronal fate determination. The biggest difficulty in analysis of Notch signaling is the random distribution of different stages of cells in the developing ventricular zone, which is thus called a salt-and-pepper pattern. In medulla neurogenesis, however, cell differentiation is well organized spatiotemporally and the developmental process of medulla neurons can be viewed as a medial-lateral array of progressively aged cells across the optic lobe. Such features allowed the functions of Notch to be precisely analyzed. Cells are classified into four types according to their developmental stages: neuroepithelial cells expressing PatJ, neuronal progenitor I expressing a low level of Dpn, neuronal progenitor II expressing L(1)sc and neuroblasts expressing high levels of Dpn. The Notch signal is activated in neuronal progenitor I and II. The EGFR signal turns on in the neuronal progenitor II stage and progresses the stage by activating L(1)sc expression. Cells become neuroblasts when the Notch and EGFR signals are shut off. Cells stay as neuronal progenitor I when Notch signal alone is activated, whereas cells stay as neuronal progenitor II when the Notch signal is activated in conjunction with the EGFR signal. Although the Notch signal is once activated, it must be turned off to let cells differentiate into neuroblasts. In neuronal progenitor II, E(spl)-C expression is induced by Notch signaling, and the increased E(spl)-C next downregulates Dl expression and subsequent activation of the Notch signal (Yasugi, 2010).

What does Notch do in medulla neurogenesis? It is infered that the Notch signal sustains cell fates, whereas the EGFR signal progresses the transitions of cell fate. This was well documented when a constitutively active form of each signal component was induced. EGFR, or its downstream Ras, induces expression of L(1)sc but does not fix its state, even though the constitutively active form is employed. At the same time, a constitutively active Notch sustains cell fates in a cell-autonomous manner. Constitutively active N receptors, by contrast, autonomously determine cell fates depending on the context: cells become neuronal progenitor I in the absence of EGFR and neuronal progenitor II in the presence of EGFR. The precocious neurogenesis caused by the impairment of Notch signaling suggests that Notch keeps cells in the progenitor state for a certain length of time in order to allow neuroepithelial cells to grow into a sufficient population. In the prospective spinal cord of chick embryo (Hammerle, 2007), the development from neural stem cells to neurons progresses rostrocaudally, during which the transition from proliferating progenitors to neurogenic progenitors is regulated by Notch signaling (Yasugi, 2010).

Although Notch plays a pivotal role in determining cell fate between neural and non-neural cells, the function may be context dependent and can be classified into three categories. (1) Classical lateral inhibition is seen in CNS formation in embryogenesis and SOP formation in Drosophila. Cells that once expressed a higher level of the Notch ligand maintain their cell states and become neuroblasts. (2) Oscillatory activations are found in early development of the mouse brain (Shimojo, 2008). Progenitor cells are not destined to either cell types. (3) An association with the proneural wave found in Drosophila medulla neurogenesis as is described in this study. The Notch signal is transiently activated only once and then shuts off in a synchronized manner. The notable difference in the outcome is the ratio of neural to non-neural cells; a small number of cells from the entire population become neuroblasts or neural stem cells in the former cases (1 and 2), whereas most of the cells become neuroblasts in the latter case (3). The differences between (1) and (2) can be ascribed at least in part to the duration of development. Hes1 expression has been shown to oscillate within a period of 2 hours in the mouse, whereas in Drosophila embryogenesis, selection of neuroblasts from neuroectodermal cells takes place within a few hours. Thus, even if Drosophila E(spl) has a half-life time equivalent to Hes1, the selection process during embryogenesis probably finishes within a cycle of the oscillation. The process of medulla neuroblast formation continues for more than 1 day, but Notch signaling is activated for a much shorter period in any given cell. This raises the possibility that E(spl)/Hes1 may have a similarly short half-life but outcome would depend on the developmental context (Yasugi, 2010).

The functions of EGFR and Notch described in this study resemble their roles in SOP formation of adult chordotonal organ development; the EGFR signal provides an inductive cue, whereas the Notch signal prevents premature SOP formation. In addition, restricted expression of rho and activation of the EGFR signal assure reiterative SOP commitment. Several neuroblasts are also sequentially differentiated from epidermal cells in adult chordotonal organs (Yasugi, 2010).

Unpaired, a ligand of the JAK/STAT pathway is expressed in lateral neuroepithelial cells and shapes an activity gradient that is higher in lateral and lower in the medial neuroepithelium. The JAK/STAT signal acts as a negative regulator of the progression of the proneural wave (Yasugi, 2008). This report has shown that activation of both EGFR and Notch signaling pathways depends on the activity of the JAK/STAT signal. The JAK/STAT signal probably acts upstream of EGFR and Notch signals in a non-autonomous fashion. These three signals coordinate and precisely regulate the formation of neuroblasts (Yasugi, 2010).

Serrate-Notch-Canoe complex mediates glial-neuroepithelial cell interactions essential during Drosophila optic lobe development

It is firmly established that neuron-glia interactions are fundamental across species for the correct establishment of a functional brain. This study found that the glia of the Drosophila larval brain display an essential non-autonomous role during the development of the optic lobe. The optic lobe develops from neuroepithelial cells that proliferate by dividing symmetrically until they switch to asymmetric/differentiative divisions generating neuroblasts. The proneural gene lethal of scute (l'sc) is transiently activated by the Epidermal Growth Factor Receptor (EGFR)/Ras signal transduction pathway at the leading edge of a proneural wave that sweeps from medial to lateral neuroepithelium promoting this switch. This process is tightly regulated by the tissue-autonomous function within the neuroepithelium of multiple signaling pathways, including EGFR/Ras and Notch. This study shows that the Notch ligand Serrate (Ser) is expressed in the glia and it forms a complex in vivo with Notch and Canoe, which colocalize at the adherens junctions of neuroepithelial cells. This complex is crucial for glial-neuroepithelial cell interactions during optic lobe development. Ser is tissue-autonomously required in the glia where it activates Notch to regulate its proliferation, and non-autonomously in the neuroepithelium where Ser induces Notch signaling to avoid the premature activation of the EGFR/Ras pathway and hence of L'sc. Interestingly, different Notch activity reporters showed very different expression patterns in the glia and in the neuroepithelium, suggesting the existence of tissue-specific factors that promote the expression of particular Notch target genes or/and a reporter response dependent on different thresholds of Notch signaling (Perez-Gomez, 2013).

Cno and its vertebrate homologues AF-6/Afadin localize at epithelial AJs where they regulate the linkage of AJs to the actin cytoskeleton by binding both actin and nectin family proteins. This study found that Cno colocalizes with Notch at the AJs of NE cells in the optic lobe proliferation centers. Notch also colocalizes with its ligand Ser, which was detected at the glia, highly accumulated at the interface between NE cells and the surrounding glia. Co-immunoprecipitation experiments indicate the formation of a Ser-Notch-Cno complex in vivo, and the mutant analysis shows the functional relevance of such a complex at the glia neuroepithelium interface. The data presented in this study support the hypothesis that Cno may be stabilizing Notch at the AJs of NE cells, favoring the binding of Ser present in the adjacent glial cells. Indeed, in cno lof both Notch and Ser distribution is affected; this alteration is accompanied by an abnormally advanced proneural wave, a reminiscent phenotype to that shown by Notch lof optic lobes and also a similar phenotype found in this work in Ser lof. The activation of Notch pathway is essential to maintain the integrity of the neuroepithelium and to allow the correct progression of the proneural wave. The results show that glial Ser is responsible of such activation, promoting the expression of the m7-nuclacZ reporter in NE cells. In fact, the reduction of glial Ser either by knocking down epithelial cno or by expressing DNSer in the glia led to a decrease in the expression of the m7-nuclacZ reporter in NE cells and to an ectopic activation of the Ras/PntP1 pathway and of L'sc. It is proposed that this may be ultimately the cause of the proneural wave advance observed in those genotypes. Thus, the activation of Notch in the neuroepithelium by glial Ser, in nomal conditions, would be essential to avoid a premature activation of the EGFR/Ras/PntP1 pathway and hence of L'sc. Indeed, Notch has been shown to downregulate different EGFR/Ras signaling pathway components such as Rhomboid (Rho), required for the processing of the EGFR ligand Spitz, in other developmental contexts in which both pathways are actively cross-talking. Therefore, Notch activity in NE cells could be contributing to inhibit Rho, restricting its presence to the transition zone where Rho is very locally expressed (Perez-Gomez, 2013).

It was observed that in a WT condition Ser is present in all surface glia (perineurial and subperineurial), as shown by the expression of CD8::GFP (SerGal4>>UAS-CD8::GFP), and Notch, as tested by different reporters, is active in this tissue and highly reduced in Ser lof in the glia. This makes sense with the existence of a Ser-Notch mediated intercellular communication between the glial cells that comprise both the perineurial and subperineurial glia. Intriguingly, the knockin down and overexpression of cno in NE cells also had a clear effect on Notch activity in the glia, a reduction and an increase, respectively. This is more challenging to explain. As the cno lof in the NE led to a high reduction of both neuroepithelial Notch and glial Ser, the easiest explanation is that an 'excess' of unbound glial Ser is degraded and this impinges on the general thresholds of glial Ser, therefore causing a general reduction in the Notch activity in this tissue. This is an interesting field to explore in detail and is left open for future investigation (Perez-Gomez, 2013).

The activity of Notch in the neuroepithelium and in medulla NBs seems controversial. For example, Notch has been shown to be active in the neuroepithelium at low/null levels or in a 'salt and pepper' patter. A weak/null activity of Notch has also been reported in NBs as well as a high activation. One possibility to conciliate all these results and apparently contradictory data is that different Notch target genes used as Notch activity reporters are, in fact, differentially activated in particular regions or tissues. The results support this proposal. Four different Notch reporters were used in this study. Whereas m7-nuclacZ was expressed throughout the neuroepithelium, Gbe+Su(H)lacZ was restricted to the transition zone, although both were expressed in medulla NBs along with mβ-CD2. In addition, mβ-CD2 was strongly activated in the glia, whereas the Gbe+Su(H)lacZ and the mδ-lacZ reporters were expressed at much lower levels at this location. Differential activation of Notch targets genes has been previously reported and tissue-specific factors could contribute to this differential expression. This is an intriguing scenario to analyze in the future. The in depth analysis of other Notch reporter genes in the developing optic lobe can contribute to further clarify this issue (Perez-Gomez, 2013).

At third larval instar during optic lobe development, Dl is highly restricted to 2-3 cells at the transition zone in the neuroepithelium, where Dl activates Notch. This work has found that the other ligand of Notch, Ser, is expressed in the surrounding glia at this larval stage and it is strongly accumulated at the interface with NE cells. Ser activates Notch in the neuroepithelium and this, in turn, would contribute to restrict the activation of the Ras-PntP1 pathway and L'sc to the transition zone. Intriguingly, it was observed that Ser preferentially activates the Notch target gene m7-nuclacZ in the neuroepithelium whereas Dl activates other Notch target genes, including Gbe+Su(H)lacZ, in the transition zone. For example, the overexpression of Dl in NE cells caused an ectopic activation throughout the neuroepithelium of Gbe+Su(H)lacZ, along with dpn that also behaves as a Notch target in other systems, and a repression of m7-nuclacZ . In addition, the lof of Ser in the glia caused a striking decrease in the expression of m7-nuclacZ in the neuroepithelium. One possibility to explain these observations is that the pool of Notch associated to the AJs and activated by glial Ser is subject of particular posttranslational modifications or/and is associated with other AJs proteins (including Cno) that somehow make Notch more receptive to Ser and able to activate specific target genes (i.e., m7). In this regard, it is interesting to note that Dl ectopically expressed in the glia (i.e., repoGal4>>UAS-Dl) was not detected at the interface with NE cells, where glial Ser is highly present in contact with Notch, but Dl was restricted to the outermost surface glia (perineurial glia). This result strongly indicates that Dl cannot bind or has very low affinity for this pool of Notch at the AJs, hence being actively degraded in the subperineurial glia. This low affinity of Dl by Notch at this location further suggests that this pool of Notch at the AJs must be endowed with particular characteristics that ultimately could alter the activity properties of Su(H), explaining in turn the distinct expression pattern of Notch targets genes. Another possibility, which is not necessarily exclusive, to explain the differential activation of the Notch reporters is that they respond to different Notch thresholds. For example, m7-nuclacZ would require very low levels of Notch activation whereas Gbe+Su(H)lacZ would require high amounts of Notch signaling in NE cells. All these questions remain open for further investigation (Perez-Gomez, 2013).

Targets of Activity

L'SC activates Enhancer of split and HLH-M5 of the Enhancer of split complex, and possibly dorsal as well (Hinz, 1994).

Protein Interactions

Extramachrochaete forms inactivating heterodimers with L'SC as it does with AC and SC (Cabrera, 1994). Daughterless is required as a dimerization partner for L'SC function (Hinz, 1994).

Classical genetics indicates that the achaete-scute gene complex (AS-C) of Drosophila promotes development of neural progenitor cells. To further analyze the function of proneural genes, the effects of Gal4-mediated expression of lethal of scute, a member of the AS-C, were studied during embryogenesis. Expression of lethal of scute forces progenitor cells of larval internal sensory organs to take on features of external sensory organs. Normally, these cells are committed to this fate independent of AS-C activity. Surprisingly, overexpression of l'sc does not result in supernumerary neural cells. Supernumerary neural cells can be induced ectopically only if daughterless is overexpressed, either alone or together with lethal of scute: cells of the amnioserosa and the hindgut then express neuronal markers. Cells of the proctodeal anlage, which normally lack neural competence, acquire the ability to develop as neuroblasts following transplantation into the neuroectoderm. Activated Notch prevents the cells of the neuroectoderm from forming extra neural tissue when they express an excess of proneural proteins. Under the present conditions, lateral inhibition is thus dominant over the activity of proneural genes (Giebel, 1997).

DEVELOPMENTAL BIOLOGY

Embryonic

Like achaete and scute, l'sc is expressed in very specific subsets of cells in neuroblasts of the ventral neurectoderm (Martin-Bermudo, 1993). Gene expression in the ventral neuroectoderm is discussed at the achaete-scute complex site.

As with achaete and scute, l'sc is expressed in every neurogenic region of the fly, including the cephalic and gnathal regions. After stage 9, l'sc is expressed in the mesectodermal, central and peripheral nervous system anlage, as well as the stomatogastric nervous system and the optic lobes (Cabrera, 1990).

In head midline structures, in particular the optic lobe and stomatogastric nervous system, there may be a late phase of EGFR signaling (as assayed by the expression of aos and activated ERK) whose significance is not yet known. EGFR signaling could be involved in modifying the inhibitory feed-back loop between neurogenic and proneural genes that exists in other neurectoderm cells. In the head midline neurectoderm, regulation of proneural and neurogenic genes has to be different. Thus, instead of a short burst of proneural gene expression in proneural clusters that is resolved into expression in individual neuroblasts, proneural genes are expressed for a long period of time; at the same time, the expression is never restricted to single neuroblasts. Since genes of the E(spl) complex are expressed in the same cells that express l’sc, the inhibitory loop between E(spl)-C and proneural genes must be interrupted at some level. It is possible that Egfr signaling is causing the interruption of this inhibitory loop. Based on genetic studies of Notch and Egfr signaling in the compound eye, it has been speculated that one of the consequences of Egfr activation (which ultimately is required for all ommatidial cell types to differentiate) is to inhibit N signaling, since constitutively active N inhibits ommatidial cell differentiation by preventing response to differentiative signals. However, the same effect could be achieved if Egfr signaling, similar to what is proposed here for the midline neurectoderm, interrupts the inhibition of proneural genes by E(spl). Although this would not prevent N signaling, it would cancel the effect of N signaling on downregulating proneural genes and thereby keep cells in a state of competency to respond to signals (Dumstrei, 1998).

The expression of the proneural gene lethal of scute is required for the development of the majority of the procephalic neuroblasts. lethal of scute expression patterns correspond to many of the identifiable 23 groups of neuroblasts in the developing brain. l'sc expression in the procephalic neurectoderm is controlled in partially overlapping domains of the neuroectoderm. Loss of function of a given head gap gene results in the absence of l'sc expression in its domain, followed by the absence of neuroblasts that would normally segregate from this domain (Younossi-Hartenstein, 1997).

Neuroblasts delaminate from the procephalic neurectoderm in a stereotyped spatiotemporal pattern that is tightly correlated with the expression of l'sc. The pattern of neuroblasts was reconstructed by using the marker asense; similar to its expression in the ventral neuroblasts, asense labels all brain neuroblasts. seven-up, expressed in specific subsets of neuroblasts making up approximately one-third of the total, is also used as a marker. For most, if not all, of these clusters the number of neuroblasts and the time of onset of svp expression are absolutely invariant (Younossi-Hartenstein, 1996).

The convergence of Notch and MAPK signaling specifies the blood progenitor fate in the mesoderm

Blood progenitors arise from a pool of pluripotential cells ('hemangioblasts') within the Drosophila embryonic mesoderm. The fact that the cardiogenic mesoderm consists of only a small number of highly stereotypically patterned cells that can be queried individually regarding their gene expression in normal and mutant embryos is one of the significant advantages that Drosophila offers to dissect the mechanism specifying the fate of these cells. This paper shows that the expression of the Notch ligand Delta (Dl) reveals segmentally reiterated mesodermal clusters ('cardiogenic clusters') that constitute the cardiogenic mesoderm. These clusters give rise to cardioblasts, blood progenitors and nephrocytes. Cardioblasts emerging from the cardiogenic clusters accumulate high levels of Dl, which is required to prevent more cells from adopting the cardioblast fate. In embryos lacking Dl function, all cells of the cardiogenic clusters become cardioblasts, and blood progenitors are lacking. Concomitant activation of the MAPK pathway by EGFR and FGFR is required for the specification and maintenance of the cardiogenic mesoderm; in addition, the spatially restricted localization of some of the FGFR ligands may be instrumental in controlling the spatial restriction of the Dl ligand to presumptive cardioblasts (Grigorian, 2011).

In this study pursued two goals: to elucidate the precise location and cellular composition of the cardiogenic mesoderm, and to analyze the mechanism by which Notch becomes activated in the restricted subset of these cells that become blood progenitors. The findings show that the cardiogenic mesoderm is comprised of segmentally reiterated pairs of clusters (cardiogenic clusters) defined by high expression levels of Dl, L'sc and activated MAPK. The MAPK pathway, activated through both EGFR and FGFR signaling, is required for the specification (EGFR) and maintenance (EGFR and FGFR) of all cardiogenic lineages. As shown previously, the default fate of all cardiogenic cells is cardioblasts. Notch activity triggered by Dl is required for the specification of blood progenitors (thoracic cardiogenic clusters) and nephrocytes (abdominal cardiogenic clusters), respectively. One of the downstream effects of MAPK signaling is to maintain high levels of Dl in the cardiogenic clusters, and to help localize Dl expression toward a dorsal subset of cells within these clusters, which will become cardioblasts. Dl stimulates Notch activity in the surrounding cells, which triggers the blood progenitor/nephrocyte fate in these cells (Grigorian, 2011).

The cardiogenic clusters form part of a larger population of mesodermal cells defined by high expression levels of l'sc. Based on l'sc in situ hybridization, these authors mapped 19 clusters of l'sc expressing cells within the somatic mesoderm. Many of these clusters (called 'myogenic clusters' in the following) give rise to one or two cells that transiently maintain high levels of l'sc, whereas the remaining cells within the cluster lose expression of l'sc. The l'sc-positive cells segregate from the mesoderm to a more superficial position, closer to the ectoderm, undergo one final mitotic division, and differentiate as muscle founder cells. Dl/Notch mediated lateral inhibition was shown to act during the singling-out of muscle founders from the myogenic clusters. Loss of this signaling pathway caused high levels of L'sc to persist in all cells of the myogenic clusters, with the result that all cells developed as muscle founders. Interestingly, loss of l'sc had only a mild effect, consisting of a slight reduction in muscle founders. This is similar to what was find in this paper in l'sc-deficient embryos, which show only a mild reduction in cardioblasts and other cardiogenic lineages (Grigorian, 2011).

The developmental fate of most of the L'sc-positive clusters within the dorsal somatic mesoderm is different from that of the ventral and lateral myogenic clusters discussed above, even though several parallels concerning the morphogenesis, proliferation, and dependence on Dl/Notch signaling are evident. The somatic (anterior, Wg-positive) mesoderm is divided into a dorsal and ventral domain based on the expression of Tin. Initially expressed at high levels in the entire mesoderm, this gene is maintained only in the dorsal mesoderm, as a result of Dpp signaling from the dorsal ectoderm. The dorsal somatic mesoderm, which is called 'early cardiogenic mesoderm', includes four L'sc-positive clusters, C2 and C14-C16. The development of C2 has been described in detail. C2 gives rise to a progenitor that divides twice; two of the progeny become the Eve-positive pericardial cells. Meanwhile, C15 which appears later at the same position as C2, seems to behave like a 'normal' myogenic cluster. It produces a progenitor that divides once and forms the founders of the dorsal muscle DA1. As shown in this paper, the two remaining dorsal clusters, C14 and C16, give rise to the cardioblasts. It is noted that the Eve-positive progenitors, as well as the cardioblasts, resemble the muscle founders derived from the typical myogenic clusters in three aspects. First, they segregate toward a superficial position, close to the ectoderm, relative to the remainder of the cells within the clusters. Secondly, they undergo one (in case of C2: two) rounds of division right after segregation. And third, they are restricted in number by Dl/Notch signaling: in all cases, they are increased in number following Dl or Notch loss of function (Grigorian, 2011).

Cardiogenic clusters, like myogenic clusters, also depend on the MAPK signaling pathway. Past studies have shown that in the Eve-positive C2 and C15 clusters, Ras, is capable of inducing the formation of additional Eve-positive progenitors. Ras is a downstream activator of both the EGFR and FGFR tyrosine kinase pathways, both of which have been seen to be important for the formation of the Eve-positive progenitors. With a loss of the FGFR pathway, Eve-positive progenitors of both the C2 and C15 cluster are lost; by contrast, the EGFR pathway affects only C15. The balanced activity of MAPK and Notch, which in part depends on reciprocal interactions between these pathways, determines the correct number of C2/C15 derived progenitors. Ras-induced MAPK activation upregulates the expression of other MAPK signaling pathway members (autoregulatory feed-back loop), but also stimulates the antagonist Argos, as well as Dl. Dl-activated Notch, in turn, inhibits MAPK signaling (Grigorian, 2011).

Both Dl/Notch and MAPK signaling are active in the C14 and C16 clusters, which constitute the definitive cardiogenic mesoderm. MAPK activity is required for the maintenance of all lineages derived from these clusters, as shown most clearly in the EGFR LOF phenotype that entails a lack of cardioblasts, blood progenitors, and pericardial nephrocytes. Overexpression of Ras results in an increased number of all three cell types, which indicates that the C14/C16 clusters attain a larger size, possibly by an additional round of mitosis. The phenotype seen in embryos suffering from loss- or overexpression of Dl/Notch pathway members can be interpreted in the framework of a classical lateral-inhibition mechanism: Dl is upregulated in the C14/C16 derived cardioblast progenitors (analogous to the Eve-progenitors of C2/C15), from where it activates Notch in the remainder of the C14/C16 cells; these cells are thereby inhibited from forming cardioblasts, and instead become nephrocytes/blood progenitors. The level of Notch activity affects the expression of tin (low Notch) and the GATA homolog srp (high Notch), which triggers the fate of cardioblasts and blood progenitors/nephrocytes, respectively (Grigorian, 2011).

MAPK is required for the initial activation of Dl in the cardiogenic clusters (just as in the myogenic clusters). Input from the pathway is most likely also instrumental in the subsequent restriction of Dl to the cardioblast progenitors. The positive interaction between MAPK and Notch signaling could occur at several levels. A mechanism shown for the ommatidial precursors of the eye disc involves Ebi and Strawberry notch (Sno), which are thought to act downstream of EGFR signaling and lead to an upregulation of Dl through the Su(H) and SMRTER complex (Grigorian, 2011).

Generally, when one progenitor cell is seen to give rise to two different cell types it is accomplished in one of two ways. One: there is an asymmetric division, where a factor expressed by the progenitor is segregated into only one daughter cell; two: a non-uniformly expressed extrinsic signal effects one cell, but not its neighboring sibling. In the posterior (abdominal) segments of the Drosophila cardiogenic mesoderm, inhibition of Notch by Numb accounts for the asymmetric activity of Notch in a small set of cardiogenic mesoderm cells, the Svp-positive cells. If Numb function is removed, these cells, which normally produce two cardioblasts and two pericardial nephrocytes, instead give rise to four cardioblasts. However, multiple nephrocytes per segment remain in numb loss-of-function mutations; furthermore, loss of numb does not cause any defect in the blood progenitors, where asymmetrically dividing Svp-positive cells are absent. This suggests that in addition to the numb-mediated mechanism, directional activation of Notch by one of its ligands is required for the majority of nephrocytes and all of the blood progenitors. It is proposed in this study that the spatially restricted upregulation/maintenance of Dl in nascent cardioblasts acts to activate Notch in the remainder of the cells within each cardiogenic cluster, which promotes their fate as blood progenitors and nephrocytes (Grigorian, 2011).

The Notch signaling pathway is typically associated with members of two different types of bHLH transcription factors. One type act as activators, while the other act as repressors. In the context of lateral inhibition, best studied in Drosophila neurogenesis, activating bHLH transcriptions factors, including genes of the AS-C like l'sc, are expressed at an early stage in clusters of ectodermal or mesodermal cells, where they activate genetic programs that promote differentiative pathways such as neurogenesis, or myogenesis/cardiogenesis. Subsequently, Notch ligands initiate the Notch pathway in these clusters; cells with high Notch activity turn on members of the Hairy/E(spl) (HES) family of bHLH genes which act as repressors and abrogate the transcriptional programs that had been set in motion by the activating bHLH factors. This paper shows that the gene cassette consisting of the Notch signaling pathway, as well as activating and repressing bHLH factors, operates in the cardiogenic mesoderm to determine the balance between cardioblasts and blood progenitors/nephrocytes. As discussed in the following, the same cassette also appears to be centrally involved in the specification of vascular endothelial cells and hematopoietic stem cells in vertebrates, which adds to the list of profound similarities between Drosophila and vertebrate blood/vascular development (Grigorian, 2011).

Even prior to the appearance of hemangioblasts, the lateral mesoderm of vertebrates is prepatterned by sequentially activated signaling pathways and transcriptional regulators similar to those that act in flies. The Wnt/Wg pathway, for example, separates subdomains of the mesoderm in vertebrates and Drosophila, as well as more ancestral ecdysozoans. Notch signaling plays an essential role in generating boundaries between segmental, as well as intra-segmental, subdomains within the ectoderm and mesoderm. The FGF signaling pathway predates the appearance of Bilaterians and plays a highly conserved role in early mesoderm patterning. Likewise, specific sets of transcriptional regulators are the targets of these signaling pathways (e.g., twist, zfh and myostatin) and play a role during the establishments of cell fate in the mesoderm. It appears, therefore, that the bilaterian ancestor featured a mesodermal subdomain, the 'cardiogenic/lateral mesoderm, in which signals of the Wg, BMP, Notch, and FGF pathways and conserved sets of transcriptional regulators established boundaries and cell fate in the mesoderm (Grigorian, 2011).

The vertebrate gene encoding an activating bHLH factor with sequence similarity to the Drosophila AS-C genes is SCL. SCL expression in the lateral mesoderm marks the first appearance of hemangioblasts; note that SCL is also expressed widely in the developing vertebrate CNS. In Zebrafish, from their site of origin in the lateral mesoderm, SCL-positive hemangioblasts migrate dorso-medially and form the intermediate cell mass (ICM). The ICM is the site of primitive endothelial blood vessel and hematopoietic cell specification. Gain of function studies carried out in zebrafish embryos have shown SCL to be one of the genes important in specifying the hemangioblast from the posterior lateral plate mesoderm. The specification of hemangioblast here comes at the expense of other mesodermal cell fates, namely the somitic paraxial mesoderm. In mice, lack of SCL affects blood and vascular development as SCL mutants are bloodless and show angiogenesis defects in the yolk sac (Grigorian, 2011).

Vertebrate homologs of the repressive Drosophila Hairy/E(spl) family of bHLH genes are the Hes and Hey (hairy/Enhancer-of-split related with YRPW motif) genes. A well studied member of the Hey family in zebrafish is gridlock, which is required for the specification of hematopoietic progenitors from the ICM. Hey 2 mutations in mice lead to severe congenital heart defects. In addition, the Hes protein plays a role in hematopoiesis as it is a positive regulator of Hematopoietic Stem Cell (HSC) expansion (Grigorian, 2011).

Genetic studies of the Notch receptors and their ligands in vertebrates support the idea that this pathway does indeed play a crucial role in the initial determination of hematopoietic stem cells. The yolk sac and the para-aortic splanchnopleura (P-Sp)/AGM (aorta gonad mesonephros) of Notch null mouse embryos lack HSCs. A similar phenotype is observed in mutants of Jagged 1, one of the Notch ligands. Notch is thought to be the deciding factor between hematopoietic and endothelial cell fates when the two originate from a common precursor or hemangioblast. In murine mutants exhibiting lower Notch1 mRNA levels, a lack of hematopoietic precursors is seen and is accompanied by an increase in the number of cells expressing endothelial cell markers. Likewise, in Drosophila, loss of Notch is associated with an increase in cardioblast number and a loss of blood precursor cells (Grigorian, 2011).

Drosophila optic lobe neuroblasts triggered by a wave of proneural gene expression that is negatively regulated by JAK/STAT

Neuroblasts (NBs) generate a variety of neuronal and glial cells in the central nervous system of the Drosophila embryo. These NBs, few in number, are selected from a field of neuroepithelial (NE) cells. In the optic lobe of the third instar larva, all NE cells of the outer optic anlage (OOA) develop into either NBs that generate the medulla neurons or lamina neuron precursors of the adult visual system. The number of lamina and medulla neurons must be precisely regulated because photoreceptor neurons project their axons directly to corresponding lamina or medulla neurons. This study shows that expression of the proneural protein Lethal of scute [L(1)sc] signals the transition of NE cells to NBs in the OOA. L(1)sc expression is transient, progressing in a synchronized and ordered 'proneural wave' that sweeps toward more lateral NEs. l(1)sc expression is sufficient to induce NBs and is necessary for timely onset of NB differentiation. Thus, proneural wave precedes and induces transition of NE cells to NBs. Unpaired (Upd), the ligand for the JAK/STAT signaling pathway, is expressed in the most lateral NE cells. JAK/STAT signaling negatively regulates proneural wave progression and controls the number of NBs in the optic lobe. These findings suggest that NBs might be balanced with the number of lamina neurons by JAK/STAT regulation of proneural wave progression, thereby providing the developmental basis for the formation of a precise topographic map in the visual center (Yasugi, 2008).

NE cells are programmed to differentiate into NBs from the medial edge of the developing optic lobe. The wave of differentiation progresses synchronously in a row of cells from medial to lateral optic lobe sweeping across the entire NE sheet; it is preceded by the transient expression of the proneural gene l(1)sc. As the NBs at the medial edge are oldest and the more lateral ones are youngest, developmental process of medulla neurons can be viewed as an array of progressively aged cells across optic lobe mediolaterally. This contrasts with NB formation in the embryonic CNS in which a small number of cells are selected from NE cells to become NBs, leaving the majority of NE cells to develop into non-neural cells. The optic lobe proneural wave is reminiscent of the morphogenetic furrow that moves across the developing eye imaginal disc. The morphogenetic furrow is the site where differentiation from neuroepithelium to photoreceptor neurons is initiated. The progression is driven by the secreted Hh expressed in the differentiated photoreceptor cells. By contrast, the proneural wave still progresses even when NB differentiation is impaired, suggesting that its progression is not driven by a factor emanating from differentiated NBs. No progression-defective phenotypes were observed when Hh or Decapentaplegic (Dpp) signaling was reduced. The model is favored that the proneural wave progression is driven by an intrinsic mechanism such as a segmentation clock and is negatively regulated by JAK/STAT pathway. As the JAK/STAT ligand Upd is expressed only by the most lateral NE cells, proliferation of the NE cells moves the source of ligand laterally and as a consequence releases more medial NE cells from negative regulation and allows the proneural wave to progress laterally. Alternatively, distribution of the Upd ligand and/or the response to Upd changes as the NE cells age as graded 10xSTAT-GFP activities are more prominent in the early stage. Non-autonomous action of JAK/STAT signal indicates that it does not directly regulate L(1)sc expression and there are second signal(s) that regulate the expression of L(1)sc under the control of JAK/STAT signal (Yasugi, 2008).

Three out of the four AS-C genes [sc, l(1)sc and ase] are expressed during medulla neurogenesis. l(1)sc is expressed in NE cells and ase in NBs, while sc is expressed both in NE cells and NBs. Deleting all AS-C genes causes as significant delay as da in NB formation but does not completely eliminate NB formation, suggesting that Da-dependent proneural gene activities are required for timely onset of NB formation. Mutation for sc or ase alone does not affect NB formation, but the simultaneous deletion of sc and l(1)sc causes the delay in NB formation and the additional deletion of ase further delays NB formation. ase expression is not altered in the absence of l(1)sc and l(1)sc is not altered in the absence of ase, indicating that l(1)sc and ase both contribute to the differentiation from NE cells to NBs. Although the contribution of Sc cannot be formally excluded, the highly specific expression pattern led to the inference that L(1)sc plays a major role in the proneural wave (Yasugi, 2008).

JAK/STAT signaling is known to regulate stem cell maintenance in the adult germline of Drosophila. In the male testis, germline stem cells (GSCs) attach to a cluster of somatic support cells at the tip (hub) of the testis. When a GSC divides, the daughter retaining contact with the hub maintains self-renewing GSC identity, while the other daughter differentiates into gonialblast. Upd is specifically expressed in the hub cells and activates JAK/STAT signal in the GSCs to maintain stem cell state. In the female ovary, JAK/STAT signaling is required in the somatic escort stem cells whose daughters encase developing cysts. This study shows that in the optic lobe development, JAK/STAT signaling maintains NE cells in an undifferentiated state. It is suggested that a common mechanism operates in both these developmental systems. Loss of Hop or Stat92E function decreases number of stem cells and ectopic expression of Upd results in over proliferation of undifferentiated cells. The cell fate may be determined by the distance of the cells from the source of ligand; the cells farther from the source commence to differentiate (Yasugi, 2008).

In the vertebrate CNS, NE cells first proliferate by symmetric cell divisions and differentiate into neurons and glia in later developmental stages. JAK/STAT signaling has been implicated in maintenance of neural precursor cells, but there is no clear evidence that those cells are in the same developmental stage as described in this study for Drosophila. Further study of JAK/STAT signaling will reveal whether a common mechanism underlies stem cell development in both Drosophila and vertebrates, and should give new insights into vertebrate CNS neurogenesis (Yasugi, 2008).

Development of a precise topographic map (retinotopic map) in Drosophila is known to involve regulation of lamina neuron development with respect to the incoming R axons. The lateral NE sheet is continuous with a groove called the lamina furrow where NE cells are arrested at G1/S phase. The arriving R axons deliver Hh and liberate the arrested NE cells to proliferate and develop into lamina neuron precursors. And, thus, R axons can induce the development of their synaptic partners in their vicinity to balance the number of R axonal termini and lamina neurons. However, medulla development does not depend on inputs from the R axons in the early phase. This study shows that both lamina and medulla neurons are derived from the continuous NE sheet. Large clones of cells mutant for the JAK/STAT signaling cause immature proliferation of medulla NBs at the expense of lamina neurons, suggesting that the number of NE cells serves as the limiting factor to generate precursors for lamina and medulla neurons. Thus, the number of medulla neurons is roughly regulated at the level of NBs whose generation might be balanced indirectly with the number of lamina neurons through regulating proneural wave progression by JAK/STAT signaling. JAK/STAT signaling therefore plays an important role in the formation of a precise retinotopic map in the visual center (Yasugi, 2008).

Notch regulates the switch from symmetric to asymmetric neural stem cell division in the Drosophila optic lobe

The proper balance between symmetric and asymmetric stem cell division is crucial both to maintain a population of stem cells and to prevent tumorous overgrowth. Neural stem cells in the Drosophila optic lobe originate within a polarised neuroepithelium, where they divide symmetrically. Neuroepithelial cells are transformed into asymmetrically dividing neuroblasts in a precisely regulated fashion. This cell fate transition is highly reminiscent of the switch from neuroepithelial cells to radial glial cells in the developing mammalian cerebral cortex. To identify the molecules that mediate the transition, neuroepithelial cells were microdissected, and their transcriptional profile was compared with similarly obtained optic lobe neuroblasts. Genes encoding members of the Notch pathway were found expressed in neuroepithelial cells. Notch mutant clones are extruded from the neuroepithelium and undergo premature neurogenesis. A wave of proneural gene expression is thought to regulate the timing of the transition from neuroepithelium to neuroblast. The proneural wave transiently suppresses Notch activity in neuroepithelial cells, and inhibition of Notch triggers the switch from symmetric, proliferative division, to asymmetric, differentiative division (Egger, 2010).

In the developing mammalian cortex, neural stem cells initially divide symmetrically to produce two neural stem cells, thereby increasing the neural precursor pool. The radial glial cells subsequently divide asymmetrically to produce a neural stem cell and either a basal progenitor cell or an immature neuron. Most basal progenitor cells divide once more to generate two postmitotic neuron. The Notch signalling pathway is thought to play a role in maintaining the undifferentiated state of neuroepithelial cells, radial glia and basal progenitors, but the downstream signalling cascades activated in these cells might be differentially regulated. Neurogenesis is initiated by proneural genes, such as Mash1 and Neurogenin2 (Ngn2) (Egger, 2010).

This study shows that this sequence of neurogenic events is remarkably similar to that seen in the development of the optic lobe in Drosophila. Notch is activated in the neuroepithelial cells, which remain undifferentiated. The proneural gene l'sc is expressed within the transition zone, and levels of Delta are increased, while Notch activity is decreased. Thus neuroepithelial cells ultimately give rise to a variety of differentiated neurons, but only after they have passed through the transition zone (Egger, 2010).

Low levels of Delta expression were found thoughout the optic lobe neuroepithelium, with increased expression in the transition zone. Several Brd genes were found within the neuroepithelium. Negative regulation of Delta activity by the Brd proteins would be expected to further reduce the level of Delta signalling. This situation might be analogous to the oscillations in Delta and Ngn2 levels observed in vertebrates, and it will be interesting to assess whether the expression of Delta, HLHm5 or proneural genes also oscillate in flies. Strikingly, when Delta activity is inhibited throughout the epithelium, the premature transformation of the entire neuroepithelium into neuroblasts is observed. This suggests that neuroepithelial cells might both send and receive the Notch signal (Egger, 2010).

Higher levels of Delta were observed in the L'sc positive transition zone. High levels of Delta or Serrate can inhibit Notch signalling through cis-inhibition, suggesting one possible mechanism for the downregulation of Notch signalling at the transition zone. Interestingly, very recent results suggest that cis-inhibition can create sharp boundaries and this could be the role of the high levels of Delta that were observe in the transition zone (Egger, 2010).

Epithelial integrity might be important to maintain proliferative cell division. This study shows that Notch mutant clones are extruded from the neuroepithelium. Furthermore, expression in the optic lobe neuroepithelial cells was found of a number of genes involved in cell adhesion. Notch could regulate cell adhesion molecules at the transcriptional level, or might itself form a complex with adhesion molecules. In either case, Notch loss of function would disrupt cell adhesion and lead to the extrusion of epithelial cells. Subtypes of cadherins, such as DE-Cad, Cad99C, Fat, which were found preferentially expressed in the neuroepithelium, might be activated by Notch to maintain the neuroepithelium, and repressed by L'sc to promote neurogenesis. Notch mutant clones also upregulate expression of the neuroblast transcription factor Dpn (but not of L'sc), and divide asymmetrically only once they have delaminated from the epithelium. In contrast to Notch mutant clones, L'sc misexpression clones upregulate Dpn and switch to asymmetric division whilst still embedded within the neuroepithelium. L'sc acts, at least in part, through repression of Notch signalling, but might also induce neuroblast-specific genes directly (Egger, 2010).

JAK/STAT signalling negatively regulates the progression of the proneural wave and neurogenesis in the optic lobe. Interestingly, the ability of Notch to maintain radial glial cell fate appears to be largely dependent on functional JAK/STAT signalling. It remains to be seen whether the Notch pathway interacts with JAK/STAT in the Drosophila optic lobe (Egger, 2010).

This study has shown that the development of the Drosophila optic lobe parallels that of the vertebrate cerebral cortex, suggesting that the pathways regulating the transition from symmetric to asymmetric division might be conserved from flies to mammals. Identifying the effector genes that are regulated by Notch and L'sc, and the links between JAK/STAT and Notch signalling, will yield further insights into the molecular mechanisms that maintain an expanding neural stem cell pool and regulate the timely transition to differentiation (Egger, 2010).

A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system

Brain areas each generate specific neuron subtypes during development. However, underlying regional variations in neurogenesis strategies and regulatory mechanisms remain poorly understood. In Drosophila, neurons in four optic lobe ganglia originate from two neuroepithelia, the outer (OPC) and inner (IPC) proliferation centers. Using genetic manipulations, this study found that one IPC neuroepithelial domain progressively transformed into migratory progenitors that matured into neural stem cells (neuroblasts) in a second domain. Progenitors emerged by an epithelial-mesenchymal transition-like mechanism that required the Snail-family member Escargot and, in subdomains, Decapentaplegic signaling. The proneural factors Lethal of scute and Asense differentially controlled progenitor supply and maturation into neuroblasts. These switched expression from Asense to a third proneural protein, Atonal. Dichaete and Tailless mediated this transition, which was essential for generating two neuron populations at defined positions. It is proposed that this neurogenesis mode is central for setting up a new proliferative zone to facilitate spatio-temporal matching of neurogenesis and connectivity across ganglia. (Apitz, 2014).

Recent studies have distinguished three neurogenesis modes in the Drosophila CNS. First, type I neuroblasts arise from neuroepithelia and generate GMCs, which produce neuronal and glial progeny. Second, Dpn+ type II neuroblasts in the dorsomedial central brain go through a transit-amplifying Dpn+, Ase+ population, called intermediate neural precursors, which generate GMCs and postmitotic offspring. Third, lateral OPC neuroepithelial cells bypass the neuroblast stage and generate lamina precursor cells (LPCs) that divide once to produce lamina neurons. The current results provide evidence for a fourth strategy: p-IPC neuroepithelial cells give rise to progenitors that migrate to a second neurogenic domain, where they mature into type I neuroblasts. These progenitors are distinct, as they originate from the neuroepithelium, do not express markers for neuroblasts, intermediate neural precursors, GMCs or postmitotic neurons, and acquire NSC properties after completing their migration (Apitz, 2014).

Migratory progenitors arise from the p-IPC by a mechanism that shares cellular and molecular characteristics with EMT. On the basis of data on gastrulation and neural crest formation, EMT is commonly associated with cells adopting a mesenchymal state, enabling them to leave their epithelial tissue and migrate through the extracellular matrix to new locations. A recent study also reported an EMT-like process in the mammalian neocortex, whereby newborn neurons and intermediate progenitors delaminate from the ventricular neuroepithelium and radially migrate to the pial surface. This study observed that neuroepithelial cells at the p-IPC margins and migratory progenitors upregulated the Snail homolog Esg, whereas E-cad levels were decreased. Moreover, esg knockdown caused the formation of ectopic E-cad-expressing clusters adjacent to the p-IPC. Although this is a previously uncharacterized role of Drosophila esg, these findings are consistent with the requirement of two Snail transcription factors, Scratch1 and 2, and downregulation of E-cad in cortical EMT migration (Apitz, 2014).

Although TGFβ signaling is well known to induce EMT, it was unclear whether it could have such a role in the brain. Two lines of evidence are consistent with a requirement of the Drosophila family member Dpp. First, it is expressed and downstream signaling is activated in dorsal and ventral p-IPC subdomains and emerging cell streams. Second, tkv mutant cells form small neuroepithelial clusters in p-IPC vicinity. Similar to the neural crest, where distinct molecular cascades control delamination in the head and trunk, region-specific regulators may also be required in p-IPC subdomains. Because neuroblasts derived from Dpp-dependent cell streams map to defined areas in the d-IPC, this pathway could potentially couple EMT and neuron subtype specification (Apitz, 2014).

Cell migration is an essential feature of vertebrate brain development. Commonly, postmitotic immature neurons migrate from their proliferation zones to distant regions, where they further differentiate and integrate into local circuits. Examples include the radial migration of projection neurons and tangential migration of interneurons in the embryonic cortex, as well as migration of interneuron precursors in the rostral migratory stream to the olfactory bulb in adults. In contrast, IPC progenitors develop into NSCs (neuroblasts) after they migrated. A recent study found that NSCs relocating from the embryonic ventral hippocampus to the dentate gyrus act as source for adult NSCs in the subgranular zone. In addition, cerebellar granule cell precursors migrate from the rhombic lip to the external granule layer, where they proliferate during early postnatal development. The migration of neural cell types that become proliferative in a new niche could therefore constitute a more general strategy. IPC progenitors form streams of elongated, closely associated cells. Despite their different developmental state, their organization is notably similar to the neuronal chain network in the lateral walls of the subventricular zone and the rostral migratory stream in mammals, or of migratory trunk neural crest cells in chick. Further studies will need to identify the determinants directing migratory progenitors into the d-IPC (Apitz, 2014).

Several constraints could shape a neurogenesis mode that requires migratory progenitors in the larval optic lobe. The OPC is located superficially and the IPC is positioned centrally. If medulla and lobula neurons arose by neuroepithelial duplications, these new populations would need to be integrated into an ancestral visual circuit consisting of lamina and lobula plate neurons. Cellular migration may therefore be a derived feature and serve as an essential spatial adjustment of the IPC to the newly added medulla. In principle, the migratory population could consist of immature neurons. However, migratory progenitors help to establish a new superficial proliferative niche, and to align OPC and d-IPC neuroblast positions. This in turn enables the OPC and IPC to use spatially matching birth order-driven neurogenesis patterns for establishing functionally coherent connections across ganglia (Apitz, 2014).

IPC progenitors were primed to mature into neuroblasts, but were prevented to do so in cell streams. Consistently, progenitors showed weak cytoplasmic Mira expression and prematurely differentiated into neuroblasts following loss of Pcl. Although Dichaete has been shown to repress ase to maintain embryonic neuroectodermal cells in an undifferentiated state, this study did not identify such a role in the IPC. Future studies are therefore required to distinguish whether this block in neuroblast maturation is released in the d-IPC by cell-intrinsic mechanisms or locally acting signals (Apitz, 2014).

The p-IPC and d-IPC consecutively expressed three proneural factors. esg-positive p-IPC neuroepithelial cells transiently expressed L'sc as they converted into progenitors. Following arrival in the d-IPC, progenitors matured into neuroblasts, which switched bHLH protein expression from Ase to Ato. This correlated with a change in cell division orientations from toward the lamina to the optic lobe surface and the generation of two lineages, distal cells and lobula plate neurons. The progression of neuroblasts through two stages is supported by the observations that progenitors solely entered the lower d-IPC, all neuroblasts were labeled with Ase in this area, and idpp reporter gene expression in a progenitor subset persisted in both lower and upper d-IPC neuroblasts and their progeny (Apitz, 2014).

Late l'sc knockdown reduced the number of d-IPC neuroblasts and both neuron classes, whereas p-IPC formation and EMT of progenitors appeared to be unaffected. This supports the idea that l'sc promotes neuroblast formation by controlling the rate of conversion and the progenitor supply. In contrast, ase loss severely decreased the amount of lower d-IPC neuroblasts and distal cells. This revealed a central role in the maturation of progenitors into neuroblasts, endowing them with the potential to proliferate and generate a specific lineage. Although these functions are the opposite of those observed in the OPC, they align with the role of a murine Ase homolog, Achaete-scute homolog 1 (Ascl1), in the embryonic telencephalon. Ase^- neuroblasts with type I proliferation patterns have not previously been described. Further underscoring the context-dependent activities of proneural bHLH factors, ato does not have the equivalent role of ase in conferring neurogenic properties to upper d-IPC neuroblasts, but acts upstream of differentiation programs controlling the projections of lobula plate neurons (Apitz, 2014).

Although Ase and Ato each regulated distinct aspects of d-IPC development, they were not required for either the transition or the extent of their expression domains. These functions were fulfilled by Dichaete and tll, whose cross-regulatory interactions were essential for the transition from Ase⁺ to Ato⁺, Dac⁺ expression. To link birth order and fate, temporal identity transcription factors are sequentially expressed by neuroblasts and inherited by GMCs and their progeny born during a given developmental window. Acting as the final two members of the OPC-specific series of temporal identity factors, Dichaete is required for Tll expression, whereas tll is sufficient, but not required, to inhibit Dichaete Although OPC and d-IPC neuroblasts shared the sequential expression of Dichaete and Tll, key differences include the fact that d-IPC progeny did not maintain Dichaete, that Tll was transiently expressed in newborn progeny of the upper d-IPC and was not maintained in older lineages, that Dichaete in the lower d-IPC was not required in its own expression domain for neurogenesis, and that Dichaete was required to activate tll, and tll to repress Dichaete and ase, as well as to independently upregulate Ato and Dac. Although the mechanisms that trigger the timing of the switch require further analysis, these observations support the notion that, in the d-IPC, Dichaete and tll do not function as temporal identity factors, but as switching factors between two sequential neuroblast stages. The vertebrate homologs of Dichaete and tll, Sox2 and Tlx, are essential for adult NSC maintenance and Sox2 positively regulates Tlx expression, suiggesting that core regulatory interactions between Dichaete and tll family members may be conserved (Apitz, 2014).

These studies uncovered molecular signatures for generating a migratory neural population by EMT and subsequent NSC development that are in part shared between the fly optic lobe and vertebrate cortical neurogenesis. The unexpected parallels suggest that ancestral gene regulatory cassettes imparting specific cellular properties may have been re-employed during vertebrate brain development. Analysis of p-IPC and d-IPC neurogenesis in the Drosophila optic lobe therefore opens new possibilities for systematically identifying genes regulating EMT, cell migration and sequential NSC specification (Apitz, 2014).

Effects of Mutation or Deletion

lethal of scute mutants may reach adulthood (Martin-Bermudo, 1993). This attests to the many redundancies or biological fail safe mechanisms created by the duplication of function in proneural genes.

klumpfuss shows genetic interactions with achaete, scute, lethal of scute and asense. l'sc is able to activate klu expression, but apparently only in the wing disc. There appears to be only a weak influence of the AS-C genes on klu expression, restricted to the wing area of the wing disc. However, the overall expression pattern of klu is largely independent of proneural genes. The assumption that SOPs enter apoptosis in klu mutants is supported by the observation of abundant cell death in other developing organs of klu mutants, like the legs. At certain bristle positions, such as that of the anterior sternopleura, klu is required during early bristle development immediately after proneural gene function, in order to allow a particular epidermal cell to develop as a SOP. It is suggested that klu is required only for initiation of bristle development, being downregulated once specification takes place (Klein, 1997).

Requirement for the Drosophila COE transcription factor Collier in formation of an embryonic muscle: transcriptional response to Notch signalling

During Drosophila embryogenesis, mesodermal cells are recruited to form a stereotyped pattern of about 30 different larval muscles per hemisegment. The formation of this pattern is initiated by the specification of a special class of myoblasts, called founder cells, that are uniquely able to fuse with neighbouring myoblasts. The COE transcription factor Collier plays a role in the formation of a single muscle (muscle DA3^[A] in the abdominal segments; DA4^[T] in the thoracic segments T2 and T3). Col expression is first observed in two promuscular clusters (in segments A1-A7), corresponding to two progenitors and then their progeny founder cells, but its transcription is maintained in only one of these four founder cells, the founder of muscle DA3^[A]. It is proposed that specification of the DA3^[A] muscle lineage requires both Col and at least one other transcription factor, supporting the hypothesis of a combinatorial code of muscle-specific gene regulation controlling the formation and diversification of individual somatic muscles (Crozatier, 1999).

Following establishment of the promuscular clusters, specification of the progenitors is controlled by lateral inhibition, a cell-cell interaction process mediated by the neurogenic genes Notch (N) and Delta (Dl)). In both N and Dl mutant embryos, promuscular Col expression is initiated normally but fails to become restricted to a single cell per cluster, similar to observations previously made for the expression of l’sc. As a consequence, a hyperplasic expression of Col is observed from stage 11. Since it is expressed in promuscular clusters and segregating muscle progenitors, l’sc has been proposed to play a role in muscle progenitor selection similar to the role of achaete and scute in neuroblast specification. However, in embryos lacking l’sc activity, selection of the Col-expressing progenitors occurs normally at stage 11 and muscle DA3^[A] forms as in wild type (Crozatier, 1999).

Changes in cell shape in the ventral neuroectoderm of Drosophila melanogaster depend on the activity of the achaete-scute complex genes

In the embryonic ventral neuroectoderm of Drosophila the proneural genes achaete, scute, and lethal of scute are expressed in clusters of cells from which the neuroblasts delaminate in a stereotyped orthogonal array. Analyses of the ventral neuroectoderm before and during delamination of the first two populations of neuroblasts show that cells in all regions of proneural gene activity change their form prior to delamination. Furthermore, the form changes in the neuroectodermal cells of embryos lacking the achaete-scute complex, of embryos mutant for the neurogenic gene Delta, and of embryos overexpressing l'sc, suggest that these genes are responsible for most of the morphological alterations observed (Stollewerk, 2000).

Almost all neuroectodermal cells are larger than the cells of the dorsal epidermal anlage (DEA). In comparison with the cells of the DEA in early stage 8 embryos the dorsoectodermal cells of mid-stage 8 embryos are clearly smaller. A comparison of the neurogenic region in early and mid-stage 8 embryos shows that the medial and intermediate regions of the ventral neuroectoderm (VNE) do not increase further in size whereas the lateral region enlarges considerably during this time. Due to these morphological changes the VNE can now be subdivided in relation to the cell sizes into three longitudinal regions on both sides of the midline: medial, intermediate, and lateral regions. In contrast to the cells of the medial and lateral regions, which now have approximately the same average values, the intermediate cells are smaller. Only 20% of all cells in the intermediate region are larger than the average, whereas 63% of the medial and 64% of the lateral cells exceed the average value. Most of the enlarged cells have a cuboidal shape. In every hemisegment the apical surfaces of two to four cells in the medial and lateral regions are very small (12-16 µm²) but expand basally to cover an area of 65-80 µm². One or two cells of this shape are also located in the intermediate regions but are smaller basally (48-58 µm²) than the medial and lateral cells. The number and position of these cells suggest that they correspond to the delaminating neuroblasts; this was confirmed by staining the embryos with anti-Hunchback antibody, an early marker for neuroblasts (Stollewerk, 2000).

During delamination of the SI neuroblasts the neuroectodermal cells gradually decrease in size, with the exception of a few cells located close to the midline. The cells that remain enlarged are either elongated perpendicularly to the midline or have a rounded appearance. Basally, between neuroectoderm and mesoderm, large round cells are located that lose contact with the apical surface at about 60% EL. On the basis of their position and arrangement, as well as the analysis of embryos stained for Hunchback, these cells can be identified as the SI neuroblasts. Before delamination of the SII neuroblasts, cells in the intermediate region of the neuroectoderm increase in size. Most of the SII neuroblasts delaminate from this region, whereas only a few neuroblasts arise from the medial region, where enlarged cells can also be detected. After delamination of the SII neuroblasts the enlarged cells shrink once again, as revealed by double staining with anti-Hunchback antibody and phalloidin. Cells in all regions of the VNE increase in size again prior to delamination of the SIII neuroblasts. Thus, the VNE of wild-type embryos becomes morphologically distinguishable from the DEA shortly before delamination of the SI neuroblasts. At this point the cells of the DEA have already divided, and about two-thirds of all cells in the medial and the lateral regions have become enlarged so that the DEA and the VNE are clearly distinguishable due to differences in cell size. In addition, almost all cells of the intermediate region increase in size prior to delamination of the SII neuroblasts. These data are at odds with claims that only the neuroblasts enlarge prior to delamination, both in grasshopper and Drosophila (Stollewerk, 2000).

Is there a correlation between the activity of the ASC genes and the observed morphological changes? The results presented indicate that the ASC genes are not the only ones responsible for the morphological changes that occur before delamination of the SI neuroblasts. Although the number of enlarged cells corresponds closely to the number of cells that express the ASC genes at this time point, the lack of the ASC does not result in all cells remaining the same size. Whereas in the medial region of the VNE of Df (1)260-1 embryos (that is, those lacking the ASC) only about 50% of the cells are smaller in size than in the wild type, and the lateral region is most strongly affected in comparison to the medial and intermediate regions. Therefore the enlargement of the neuroectodermal cells depends to a varying degree on the activity of the ASC genes and is additionally influenced by other factors. However, a clear correlation can be seen prior to delamination of the SII neuroblasts. At this time almost all cells of the intermediate region increase in size, which coincides with the expression of l'sc in this region. Furthermore, analysis of the VNE of embryos lacking the ASC reveals that the intermediate cells do not become enlarged prior to delamination of the SII neuroblasts, suggesting that the observed morphological changes are due to the activity of the ASC genes at this point. In addition, the shrinkage of the cells that had enlarged during delamination of the SI and SII neuroblasts is correlated with the decrease in ASC gene expression in the VNE at these time points (Stollewerk, 2000).

Analysis of wild-type and Delta mutant embryos also suggests that the ASC genes are important for the maintenance of the morphology of the neuroectodermal cells. Despite the fact that the total area of the intermediate region does not change significantly between early and mid-stage 8, cell size changes can be detected in this region shortly before delamination of the SI neuroblasts. While 20% of the intermediate cells remain larger than the average, the cells that had an average cell size in the VNE of early stage 8 embryos now split into groups of smaller cells. The fact that the number of cells that remain larger than the average corresponds to the number of cells that express the ASC genes in the intermediate region suggests that the proneural genes are required to keep these cells enlarged. This view is confirmed by analyses of the VNE of Delta mutant embryos. In Delta mutant embryos all cells of a proneural cluster continue to express the proneural genes and become neuroblasts. This altered gene expression causes all cells of a proneural cluster to remain enlarged until proneural gene expression is turned off (Stollewerk, 2000).

A correlation between increase in cell size and ASC gene expression has also been shown by the analysis of embryos labeled for ac protein and embryos overexpressing l'sc. Area measurements reveal that 85% of all cells that express ac are enlarged in these embryos. The fact that not all ac-expressing cells are larger than the average at the time point analyzed may be due to the rapidity of the morphological changes (enlargement and shrinkage) that occur immediately before and during delamination of the neuroblasts. A clear influence of a proneural gene on the cell sizes in the VNE can be seen in embryos overexpressing l'sc: 45% more cells become enlarged in the intermediate region in comparison to the wild type. Only a minor increase in the numbers of enlarged cells can be seen in the medial and lateral regions, because two-thirds of these cells already express proneural genes. In addition, the high proneural gene activity in the VNE of embryos overexpressing l'sc causes the future neuroblasts to change their morphologies: they expand not only their basal but also their apical surfaces. These data clearly show that the ASC genes have an influence on the morphologies of the neuroectodermal cells (Stollewerk, 2000).

A key role of Pox meso in somatic myogenesis of Drosophila: Poxm and l(1)sc exhibit partially redundant functions during muscle development

The Pax gene Pox meso (Poxm) was the first and so far only gene whose initial expression was shown to occur specifically in the anlage of the somatic mesoderm, yet its role in somatic myogenesis remained unknown. This study shows that it is one of the crucial genes regulating the development of the larval body wall muscles in Drosophila. It has two distinct functions expressed during different phases of myogenesis. The early function, partially redundant with the function of lethal of scute [l(1)sc], demarcates the 'Poxm competence domain', a domain of competence for ventral and lateral muscle development and for the determination of at least some adult muscle precursor cells. The late function is a muscle identity function, required for the specification of muscles DT1, VA1, VA2 and VA3. These results led to a reinterpretation of the roles of l(1)sc and twist in myogenesis and to the proposal of a solution of the 'l(1)sc conundrum' (Duan, 2007).

The development of the complex pattern of the larval body wall muscles of Drosophila provides an excellent paradigm of how a final pattern is established through precise genetic control. Each of the abdominal hemisegments A2-A7 has 30 identifiable individual muscles that develop from the somatic mesoderm. This process is initiated when the invaginated mesoderm migrates dorsolaterally under the ectoderm and is prepatterned by the segmentation genes: the product of sloppy paired (slp), whose activity is maintained by the ectodermal Wingless (Wg) signal, restricts high levels of the bHLH transcription factor Twist (Twi) to the mesodermal regions below the posterior portions of the ectodermal parasegments. These high levels of Twi function as a myogenic switch, separating the posterior somatic and cardiac mesoderm from the anterior visceral mesoderm and fat body. When the dorsal migration of the mesoderm is complete, these metamerically repeated Slp or high Twi domains are further subdivided along the dorsoventral axis by the ectodermal signal Dpp. This signal restricts transcription of tinman (tin) to the dorsal mesoderm, where its homeodomain protein specifies heart and dorsal somatic mesoderm. However, the determinant of the non-dorsal somatic mesoderm remains largely unknown. It appears that Pox meso (Poxm) expression is restricted to the ventral part of the high Twi domain by Dpp (Staehling-Hampton, 1994) to define the lateral and ventral somatic mesoderm anlage. The characterization of the role of Poxm in somatic myogenesis is therefore expected to fill an important gap in understanding of the gene network regulating this process (Duan, 2007).

Soon after this subdivision of the mesoderm, the proneural gene lethal of scute [l(1)sc] begins to be expressed in at least 19 promuscular clusters of cells within the high Twi domain. From these clusters, muscle progenitors are singled out by lateral inhibition through Notch (N) and Ras signaling and are specified by the expression of muscle-identity genes. Cells not singled out begin to express the zinc finger protein Lame duck (Lmd; also known as Minc), which specifies them as fusion-competent myoblasts (FCMs). The progenitors divide to generate different muscle founders, a muscle founder and an adult muscle precursor, or a founder and a cell producing either two adult muscle precursors or two pericardial cells. Each founder forms an individual syncytial muscle precursor by fusing with neighboring FCMs. One of the key steps in muscle pattern formation is the specification of a muscle founder by the expression of a specific set of muscle identity genes. Although an increasing number of these genes have been identified in recent years, the mechanisms that activate their transcription are still poorly understood. Hence, it is important to identify the genes whose products directly regulate the muscle identity genes (Duan, 2007).

This study describes the functional characterization of the Poxm gene. Poxm belongs to the Pax gene family whose members encode transcription factors, including a paired domain. The temporal and spatial expression patterns of Poxm and its loss- and gain-of-function phenotypes reported in this study demonstrate that it is required for most ventral and lateral abdominal muscles to develop properly in all segments and for the activation of muscle identity genes. In addition, Poxm acts itself as muscle identity gene in a few muscles and thus plays a dual role in somatic myogenesis (Duan, 2007).

Since Poxm is expressed during early myogenesis in cells that later give rise to progenitors of most of the ventral and lateral muscles, it may play an important role in the initiation of muscle patterning. To investigate which part of the Poxm^R361 muscle phenotype results from the loss of this early Poxm function, a transgene expressing Poxm only during the early myogenic stages, um1-2-Poxm, was introduced into Poxm^R361 embryos. In these embryos, the phenotypes of muscles VO4-6, VL2-VL4, VO2, VO1, LO1, LT4 and VT1 are efficiently rescued. The only muscles affected in Poxm mutants that are only slightly rescued by early Poxm are DT1, DO3 and VA1-3, in which Poxm is also expressed during later stages in their founders and/or muscle precursors. These results strongly suggest that Poxm exerts an early function, demarcating a mesodermal domain of competence for ventral, lateral and dorsolateral somatic muscle development (Duan, 2007).

The partial penetrance of the Poxm muscle phenotype suggests that the early Poxm function is largely redundant with that of other genes, an argument also raised to explain the weak muscle phenotype of l(1)sc mutants. The l(1)sc gene encodes a bHLH transcription factor the function of which is thought to be required for the selection of muscle progenitors. Therefore, the effect was examined of Poxm and l(1)sc mutations on larval muscle development in single and double mutant embryos (Duan, 2007).

In agreement with earlier studies, l(1)sc mutants exhibit a weak muscle phenotype, which deviates only slightly from that of wild-type embryos. Although Poxm^R361 embryos show a considerably stronger muscle phenotype, most lateral and dorsal muscles are normal. Assuming that Poxm and l(1)sc act independently in muscle development, it was expected that the probability of a muscle being wild-type in Df(1) l(1)sc¹⁹/Y; Poxm^R361 embryos is the product of the probabilities of the muscle being wild-type in the single mutants. Conversely, if significantly enhanced probabilities are found for muscle defects in double mutants, it may be concluded that Poxm and l(1)sc exhibit partially redundant functions during muscle development. Applying this test, it was found that most muscles are affected independently or nearly independently, with some notable exceptions. These concern muscles VL1-3, SBM, VO1, VO2, DT1, LT3, LT4 and VA3 that are more often absent. Some muscles are strongly affected in Poxm null mutants, such as muscles VO4-6 or muscles VA1-3. Among the other muscles, the more ventral and the more posterior a muscle is located within a segment, the more probable it is that it will show an enhanced phenotype in double mutants. Clearly, there is some redundancy between Poxm and l(1)sc functions in the somatic mesoderm, which is restricted largely to ventral and posterior muscles (Duan, 2007).

In Poxm mutants, only muscle DO3 is frequently duplicated. This duplication results from the transformation of muscle DT1 to DO3, as previously observed for muscles derived from the same progenitor in the absence of a muscle identity gene that is asymmetrically expressed in the two founders and muscle precursors. Thus, late expression of Poxm in the precursor of muscle DT1, but not of DO3, is crucial for their distinction and hence serves a muscle identity function. However, a more detailed analysis shows that muscle DT1 is missing in only two thirds (23/34) of all cases in which muscle DO3 is duplicated. In the remaining 11 cases, muscle DT1 is normal (4), abnormal (6) or duplicated (1). This finding suggests that the late Poxm function is necessary in about 10% (11/108) of all cases to prevent an additional division that generates a second founder of muscle DO3. Absence of Poxm in their founders results in abnormal muscles VA1-3 that cannot be rescued by the early Poxm function, which suggests that their proper specification also depends on the late function of Poxm (Duan, 2007).

These results have demonstrated that the development of larval body wall muscles depends on distinct Poxm functions during two phases. The early function of Poxm specifies, within the high Twi or Slp domain, a subdomain of competence for lateral and ventral muscle development, the 'Poxm competence domain'. This function appears to be analogous to that of tin, which specifies competence for heart and dorsal muscle development in the complementary part of the Slp domain. Poxm and tin thus subdivide the posterior Slp domain into ventral and dorsal subdomains in a manner similar to the partitioning by serpent and bap of the anterior Eve domain into the ventral fat body and the dorsal visceral mesoderm anlagen. After selection of muscle progenitors, proper development of a few muscles still depends on Poxm, which is expressed in muscles DT1 and VA1-3. This late function of Poxm participates in founder specification and muscle differentiation, as is characteristic for muscle identity genes. Finally, the findings suggest a solution to a conceptual problem of the current model of somatic myogenesis, the l(1)sc conundrum (Duan, 2007).

The muscle phenotype of Poxm mutant embryos and its rescue by early Poxm expression shows that the early Poxm function is crucial for the proper development of many ventral and lateral muscles. In addition, the generation of ectopic dorsal and dorsolateral muscles by ectopic Poxm suggests that Poxm has the ability to change cell fates and render cells competent for myogenesis. Therefore, it is proposed that early Poxm demarcates a ventral and lateral domain of competence for somatic myogenesis (Duan, 2007).

The partial penetrance of the Poxm mutant phenotype implies the existence of other competence domain genes performing partially redundant functions. Poxm and L(1)sc partially co-localize in the promuscular clusters and muscle progenitors. In addition, a detailed analysis of l(1)sc and Poxm single and double mutants demonstrates that their functions are partially redundant. Since the muscle phenotype of l(1)sc; Poxm double mutants still shows partial penetrance, additional competence domain genes should be expressed in the Slp domain. One of them is probably tin, which is initially expressed in the entire early mesoderm, because tin mutants affect muscle development in the dorsal as well as lateral and ventral Slp domain. Another candidate is D-six4, which is required for the development of specific muscles that arise from the dorsolateral and ventral regions (Duan, 2007).

Thus, after the initial subdivision of the mesoderm, the high Twi domain is further subdivided by competence domain genes, which specify domains that become competent to select progenitors of distinct subsets of somatic muscles and/or of myocardial and pericardial cells by responding to spatially restricted extracellular signals. These competence domain genes act in a cooperative manner to determine the identities of specific muscles by regulating the expression of the muscle identity genes. When one of them is inactivated, in some cells active competence domain genes can partially compensate for the inactive gene by activating its target genes such that these sometimes, but not always, exceed the threshold levels required for normal development. Hence, muscles derived from these cells exhibit a mutant phenotype with partial penetrance. For other cells, active competence domain genes can compensate completely for the missing gene activity such that these cells will adopt the proper fate and the muscles develop normally. This illustrates that competence is not a matter of 'all' or 'nothing' for muscle development. The deeper reason for this is thought to be that the genetic program regulating myogenesis is not organized in a hierarchical fashion but rather as a complex gene networkthat has an integrated function which is much more stable against mutations within the network than a hierarchical regulation would be (Duan, 2007).

Muscle identity genes usually encode transcription factors, such as Slou, Nau, Ap, Vg, Kr, Eve, Msh, Lb, Run and Kn, that are expressed in subsets of muscle progenitors and founders and determine in a combinatorial fashion the identity of each muscle founder and its subsequent differentiation into a specific muscle of defined size, shape, attachment sites, and innervation. It is envisioned the activation of these genes in promuscular clusters or, after lateral inhibition, in muscle progenitors by Twi and/or the products of competence domain genes and through combinations of localized extracellular signals from the ectoderm and mesoderm. During asymmetric division of progenitors, expression of a muscle identity gene may be maintained in one or both of the two sibling founders, or it may persist in the founder when division generates a founder and an adult muscle precursor. Late expression of Poxm illustrates all three cases. It is expressed in progenitors P26/27 and P29/VaP, which are derived from promuscular cluster 10 and give rise to the founders of muscles VA1 (F26) and VA2 (F27), and to the founder of muscle VA3 (F29) and the ventral adult precursor VaP. Poxm is also expressed in the progenitor derived from cluster 13, P11/18, which generates the founders of muscles DO3 (F11) and DT1 (F18). Although Poxm expression persists in F29 and F18 but not in their siblings, it is maintained in both sibling founders F26 and F27 (Duan, 2007).

The late function of Poxm is identified as a muscle identity function by the high correlation between absence of muscle DT1 and corresponding duplication of muscle DO3 in Poxm mutants. If Poxm was the sole determinant discriminating between F11 and F18, mesodermal ubiquitous expression of Poxm would be expected to transform muscle DO3 into DT1. The results confirm the presence of additional muscles in the region of muscle DT1. It is possible that one of these originates from a transformed F11, but it is impossible to tell whether muscle DO3 is missing because additional muscles have been recruited (Duan, 2007).

It has been shown that in the process of muscle diversification, identity genes may repress or activate other identity genes in progenitors and founders. This study found that the muscle identity gene slou fails to be activated in P11/18 of Poxm mutants. The simplest explanation of this result is that activation and maintenance of slou expression depend on Poxm in P11/18 and its offspring founders. In addition, slou expression is not maintained in F27 of Poxm mutants despite its initial activation in P26/27. It therefore appears that in P26/27 and its offspring F26 and F27, in addition to Kruppel (Kr), Poxm is necessary for the maintenance of slou expression. Although Poxm expression is maintained in both F26 and F27, slou expression is restricted to F27 because Kr is repressed in F26 by N signaling. Apparently, Kr is the crucial determinant that distinguishes F26 from F27, as F27 is altered to F26 in Kr or numb mutants (Duan, 2007).

Since Poxm is expressed in both F26 and F27, whereas its expression is restricted to F18 and not maintained in F11, its late expression in F26 and F27 must be regulated differently from that in F11 and F18 where it appears to be subject to asymmetric N signaling repressing Poxm in F11 (Duan, 2007).

These considerations imply that slou is part of the same gene network as Poxm, a conclusion consistent with the proposed gene network hypothesis since, in the first test of this hypothesis, slou had been isolated as a PRD 9 gene on the basis of its homology to the prd gene (Duan, 2007).

The mechanism of progenitor selection from the somatic mesoderm depends on a process of lateral inhibition very similar to that of neuroblast or sensory organ precursor (SOP) selection in the neuroectoderm from proneural clusters expressing the proneural genes. Because of this similarity, a search among proneural genes for 'promuscular' genes expressed in the somatic mesoderm was performed. This search identified a single proneural gene, l(1)sc, a member of the achaete-scute complex (AS-C), that is expressed in promuscular clusters of the somatic mesoderm. It was, therefore, attractive to consider its function in myogenesis to be analogous to that of proneural genes in neurogenesis. However, whereas proneural genes confer on neuroectodermal cells the ability to become neural precursors rather than epidermal cells, which is their default fate, l(1)sc does not seem to confer on mesodermal cells the ability to undergo somatic myogenesis instead of becoming part of the visceral mesoderm, heart or fat body. When L(1)sc was expressed in the entire mesoderm from stage 8 onward, other mesodermal tissues could not be transformed into somatic mesoderm, whereas a deficiency of l(1)sc resulted in only minor defects of somatic muscle development. In addition, as the l(1)sc muscle mutant phenotype can be rescued by ubiquitous mesodermal L(1)sc expression, its expression in clusters is not decisive for the formation of promuscular clusters and, therefore, l(1)sc cannot play the decisive role in the development of larval body wall muscles that has been proposed. Thus, although l(1)sc serves as an excellent marker for promuscular clusters, it lacks a property expected to be crucial for a promuscular gene. Are there genes that might qualify as promuscular genes and thus extend the close evolutionary relationship of progenitor selection between myogenesis and neurogenesis (Duan, 2007)?

There is indeed a gene that is homologous to proneural genes and expressed in the somatic mesoderm, in the absence of which somatic myogenesis is seriously disturbed. This gene is twi, whose function at stages 10 and 11 more closely corresponds to that of a promuscular gene and which, like l(1)sc, encodes a bHLH transcription factor. Although Twi is also expressed earlier when it is required for mesoderm specification during gastrulation, this early function can be distinguished from its later 'promuscular' function in temperature-sensitive mutants. In these mutants, only high levels of Twi activity, necessary for the formation of the somatic mesoderm, are abolished and no normal somatic muscles develop. Moreover, ubiquitous expression of high levels of Twi in the mesoderm blocks all other mesodermal fates, transforming them to somatic mesoderm. Since the subsequent patterning of somatic muscles depends critically on the relative levels of the products of twi and the proneural gene da, it seems appropriate to consider them both as promuscular genes (Duan, 2007).

In addition to its strict requirement for somatic myogenesis, the proposed promuscular function of twi may be subject to lateral inhibition by N signaling, in further analogy to proneural functions in neurogenesis. This is apparent from experiments demonstrating that the restriction of high Twi levels to the Slp domain during stage 9 depends on N signaling, which downregulates twi in the mesoderm underlying the anterior regions of parasegments where Slp does not override it. Since this process acts directly on an identified twi enhancer during stages 9 and 10, it is conceivable that this enhancer also responds to N signaling during the subsequent lateral inhibition. An alternative, though not mutually exclusive, mechanism for the downregulation of twi implicates the Gli-related zinc finger transcription factor Lmd (Minc), whose expression is maintained by N signaling and in the absence of which twi is not downregulated in fusion-competent myoblasts (Duan, 2007).

During lateral inhibition, loss of Twi precedes that of L(1)sc in the promuscular clusters. It is therefore possible that l(1)sc expression in these cells also depends on high levels of Twi, i.e. on Twi homodimers. Consistent with this interpretation, shifting the equilibrium between Twi homodimers and Twi-Da heterodimers in favor of the latter represses l(1)sc. Since early Poxm expression also depends on Twi, Poxm would be similarly repressed in promuscular clusters through lateral inhibition, either indirectly by repression of twi and/or directly by Twi/Da heterodimers. Such a mechanism might apply generally to both competence domain genes and muscle identity genes during lateral inhibition of promuscular clusters (Duan, 2007).

Thus, twi satisfies two criteria considered to be crucial for a promuscular gene in analogy to those of proneural genes in neurogenesis. However, a third criterion is not fulfilled by twi: its expression, in contrast to that of proneural genes in the neuroectoderm, is ubiquitous rather than restricted to promuscular clusters although this criterion is not a crucial property of proneural genes. Yet promuscular clusters from which the myogenic progenitors are selected exist, as evident from the pattern of l(1)sc expression. These promuscular clusters depend on combinations of the long-range ectodermal signals Wg and Dpp and the localized activities of the EGF signal Spi in the mesoderm and the FGF signals Pyr and Ths in the ectoderm. These signals, together with Twi and/or products of competence domain genes depending on Twi, determine the promuscular clusters by activating specific combinations of muscle identity genes. The identity of the promuscular clusters depends not only on the combination of these signals but, in the case of MAPK signaling elicited by FGF and/or EGF, also on their intensity. In addition, multiple positive and negative feedback loops of the coupled MAPK and N signaling networks ensure a stable selection and specification of muscle progenitors not only within, but also beyond, the limits of a promuscular cluster. Such a conclusion implies that these clusters are not a priori determined, but depend on the range and intensities of the MAPK activating signals, in agreement with the assumption that it is not the expression of l(1)sc that determines the promuscular clusters. In fact, it may be the absence of such a priori determined clusters of equivalent cells in the somatic mesoderm that necessitates such a complex N and Ras signaling circuitry (Duan, 2007).

Therefore, it is proposed that twi and da, instead of l(1)sc, function as promuscular genes by regulating the activities of competence domain genes, which in turn regulate the combinatorial activities of muscle identity genes and thereby specify the fates of muscle progenitors and founders. It is nevertheless surprising that l(1)sc appears to be expressed in all promuscular clusters even though its function is not necessary in most of them. It is possible that this expression pattern is an evolutionary remnant of an atavistic promuscular function of l(1)sc that was later replaced by the promuscular function of twi on whose expression l(1)sc activity depends (Duan, 2007).

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date revised: 25 March 2015
 

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